Nanotechnology and enzyme immobilization

Pseudomonas aeruginosa BTS-2 produced lipase (total yield, 55.4 U/L) optimally on a mineral based (MB) broth, supplemented with 1% (v/v) cottonseed oil as sole carbon source, after incubation at 56°C for 48 h. The presence of 4% (w/v) yeast extract in the MB broth further increased the lipase production by ~ 3-folds. The purified lipase was efficiently (~80%) immobilized in alginate than carrageenan beads. The alginate-immobilized lipase (beads) showed optima at pH 7.5 and 55°C temperature. An increase in C-chain of alcohols increased the activity of entraped lipase. Exposure of alginate beads to decyl (C:10) and dodecyl (C:12) alcohol enhanced the lipase activity by 47 and 64%, respectively. Whereas, most alkanes decreased the activity of immobilized lipase but exposure to nonane enhanced the activity by 52%. The lipase efficiently esterified ethanol and acetic acid to ethyl acetate.

The subject of the scientific research of the doctoral dissertation is the immobilization of two industrially important enzymes, protease from Bacillus licheniformis (commercial name alcalase) and peroxidase from horseradish, as well as their application in protein hydrolysis and removal of pollutants, such as phenols and synthetic dyes, from wastewater. The focus is on the development of techniques for obtaining hydrogel particles based on alginate and alginate/magnetite, as well as enzyme immobilization techniques that would enable obtaining biocatalysts with improved catalytic properties. Enzymatic hydrolysis of proteins and oxido-reduction of anthraquinone dyes catalyzed by enzymes from diverse groups we have chosen as two model reactions for evaluating the catalytic properties of immobilized biocatalysts, due to their industrial importance in the food industry and in the processes of treating wastewater from the textile industry. The development of immobilized enzymes enabled the application of enzymes in those areas where the application of free enzymes is limited due to low stability due to degradation by autocatalysis, as in the case of proteases in the case of protein hydrolysis, or due to inhibition by excess substrate, as in the case of peroxidases for the degradation of anthra-quinone dyes in the presence of hydrogen-peroxide. By immobilizing enzymes, we could obtain stable biocatalysts, which we separated from the reaction mixture after the end of the reaction by filtration or centrifugation and used repeatedly in batches or over a longer period in continuous processes. One of the most crucial factors in enzyme immobilization is the choice and design of the immobilization support because the physical and chemical characteristics of the support determine the activity of the immobilized enzyme. Inorganic supports such as iron oxide can have advantages compared to other mate-rials due to high thermal and mechanical resistance, reduced microbiological contamination, and magnetic properties, which is especially important for immobilized enzymes, where the main advantage compared to native enzymes has eased separation from reaction mix-tures and their reuse. However, due to the small number of functional groups on these inorganic supports, enough enzymes cannot be bound, nor can we reward a favorable micro-environment for the enzyme coupling, which would lead to the improvement of its catalytic properties. Therefore, by coating the magnetic particles with an additional coating of an organic polymer with a larger number of functional groups, such as alginate, a larger number of enzymes can potentially be bound, and we could achieve improved binding. In this dissertation, we examined the possibility of using alginate particles and magnetic particles coated with alginate as carriers for the immobilization of alcalase and horseradish peroxi-dase. The technique of obtaining particles under the influence of an electric field, which in the literature is often called electrostatic extrusion and which is based on the application of an electrostatic force to a suspension of polymers and biocatalysts that appears on the tip of a needle, in the form of drops, during which the drop splits into several small charged droplets, has a great possibility of application for obtaining particles with small diameters of uniform sizes on an industrial scale. Researchers have been extensively using this technique for the immobilization of various biocatalysts. However, this dissertation examined a new procedure for obtaining alginate submicron particles, which is based on a combination of ultrasonic dispersal, drying with hot air, and gelling in a column with wetted walls. Namely, we obtained alginate particles by the method of ultrasonic dispersion of alginate solution, whereby the air carrying the dispersed polymer-aerosol particles, depending on the tem-perature and unsaturation, to a greater or lesser extent encourages the evaporation of water from them and thus reduced their size, and thus obtained the nanoparticles gel in a column with wetted walls. We assessed the nanoparticles obtained in this way as carriers for the immobilization of alcalase and horseradish peroxidase and their catalytic properties with the immobilized systems with beads obtained by electrostatic extrusion. We used beads with the best characteristics (dimensions, sphericity, size distribution, magnetic properties, and magnetite-alginate ratio) for enzyme immobilization. Optimization of covalent immobilization of alcalase enzyme on alginate particles and magnetite/alginate particles obtained by electrostatic extrusion and ultrasonic spraying represents the second phase of the experiments. We have investigated the influence of im-mobilization conditions such as pH value, TRIS buffer molarity, amount of added alcalase, method of chemical activation of functional groups of particles and amount of chemical agent for activation, as well as the contact time of the enzyme with activated particles on activity, mass of bound enzyme and yield of activity. The amount of bound alcalase and peroxidase was determined by standard methods for protein determination, the Bradford method, while the proteolytic activity was determined spectrophotometrically by monitoring the hydrolysis of the chromogenic derivative of casein, azocasein. We analyzed the morphological characteristics of the obtained particles with and without immobilized enzymes using scanning electron microscopy (SEM). The size of the particles, as well as the surface morphology of the magnetic particles with and without alginate coating, were determined. To better understand the nature of the potentially established bonds between the enzyme and the carrier, we have characterized the particles of the carrier and the immobilizer by Infrared Spectroscopy with Fourier transformations. Next, we examined the particle size distribution and zeta potential using the dynamic light scattering method. We examined the magnetic properties of the particles and the strength of the coercive field using SQUID magnetometry (superconducting quantum interferometer). Also, we have compared the magnetic properties of the particles based on hysteresis curves for magnetic and magnetite/alginate particles at temperatures of 5 K and 300 K. The activity of free and immobilized alcalase we monitored in the reaction of protein hydrolysis from soy isolate and egg white under industrially relevant conditions, i.e., in a reactor with intensive mixing and in the presence of impurities based on the measurement of calcium hydroxide consumption to maintain a constant pH value during hydrolysis. We used trinitrobenzenesulfonic acid during hydrolysis, the so-called TNBS method. Kinetics of the reaction with native and immobilized enzyme, we have examined by monitoring the reaction rate of protein hydrolysis from soybean and egg white isolates at different initial substrate concentrations in initial conditions, while kinetic parameters were determined us-ing the Enzyme Kinetics application of the OriginPro software package. We evaluated the stability of the immobilized alcalase, the possibility of using it through several cycles of performing the protein hydrolysis reaction from soy and egg white isolates in a batch reactor with mechanical mixing, and the potential retention of activity after a certain storage time. We used the obtained results in the further development of the immobilized system with horseradish peroxidase. We used beads with the best characteristics (dimensions, sphericity, size distribution, magnetic properties, and ratio of magnetite and alginate) for horseradish peroxidase immobilization. The amount of bound peroxidase and its activity, as well as the activity of free peroxidase, were determined by the spectrophotometric method with pyrogallol as a substrate. We have examined the effects of immobilization time, mass ratio of enzyme and carrier, concentration of the activating agent on the mass of the bound enzyme, and the specific activity of the obtained biocatalyst on the activity. With the stability of the immobilized alcalase, we evaluated the possibility of using it through several cycles of oxidation for two anthraquinone dyes, Acid Violet 109 and Acid Blue 225. The effectiveness of immobilized peroxidases we compared based on the obtained kinetic parameters, using the Enzyme Kinetics application, in the oxidation reaction of AV109 and AB225. The work presented here has demonstrated a novel, simple, and fast technique for alginate submicron bead production by employing the custom-made ultrasonic spray atomi-zation system and compared it with the electrostatic extrusion in the hydrolytic reactions with standard and industrial food protein substrates. For these purposes, we have effectively covalently immobilized alcalase onto calcium-alginate microbeads and submicron beads after surface modification by carbodiimide. The optimal conditions for the alcalase immobilization on the alginate microbeads activated with EDAC were 5,32 IU of alcalase, 50 mM Tris-HCl buffer pH 8,5, 30 min of activation by EDAC/beads mass ratio (10 mg/0,5 g), and 20 h immobilization time. The highest activity was exhibited with alcalase immobi-lized onto UAD 0,4% (w/v) alginate beads, with the maximal value of 2716,1 IU/g support and the highest enzyme loading of 671,6±4 mg/g on the carrier in comparison to 2600,8 IU/g and 592,3±6,7 mg/g for EE biocatalyst made from 2% (w/v) alginate solution. The immobilized enzyme onto UAD 0,4% (w/v) alginate beads provided better performance than micro-sized alginate counterparts in terms of egg white and soy protein hydrolysis. We have reused optimal biocatalyst in seven successive soy protein hydrolysis cycles with a slight decrease in activity. Therefore, there is potential to use this type of alginate submicron beads obtained by novel ultrasonic spray atomization technique for enzyme immobilization. HR...

The biotechnological route to manufacturing geraniol propionate may present a feasible solution to drawbacks associated with the production of such ester by the chemical synthesis or extraction from plants. The use of such technique can be advantageous considering the ever increasing demands for such products while reducing waste production and simplifying the manufacturing process. The properties and morphology of the developed Rhizomucor miehei lipase (RML) immobilized onto activated chitosan-graphene oxide (CS/GO) support were characterized using field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA). The morphological evaluations strongly indicated successful covalent attachment of the RML on the support. It was evident from the thermogram of TGA that 13.5% of RML was successfully immobilized onto the CS/GO matrix. The approach of response surface methodology...

Urease (EC 3.5.1.5) of high activity was obtained when the enzyme was immobilized on vermiculite crosslinked with 2.5% glutaraldehyde in chilled EDTA-phosphate buffer (pH 5.5). The highest activity of the immobilized enzyme was at 65°C and pH 6.5 while the optimum temperature for free urease was found to be 25°C. The thermal stability of immobilized urease was observed to be much better than that of the free urease. When stored at 4°C, urease immobilized on vermiculite retained 69 to 81% of its activity after 60 days and 61 to 75% of its original activity was retained after 4 repeated uses.

Amberlite MB-1 was used to immobilize urease (EC.3.5.1.5). The thermal stability of the immobilized urease was better than that of the free urease. Its highest activity was obtained at 75°C and at pH 6.5 while the optimum temperature for the free urease was found to be 25°C. Urease immobilized on Amberlite MB-1 retained 65% of the original activity after 5 repeated uses and 62% of the activity after 60 days when stored at 4°C.

In this study, the intracellular lipases of Candida catenulata were examined as a whole-cell biocatalyst (WCB) of soybean oil transesterification and obtained a fatty acid methyl ester (FAME) yield of 79.1%. To facilitate the biocatalyst recovery and improve its stability, the WCB was attached to magnetic Fe 3 O 4 nanoparticles activated by a linear structure of polyethyleneimine (PEI) as the multi-amino-functionalized arm. The preparation procedure of the magnetic whole-cell biocatalysts (MWCBs) was optimized by considering the pH during immobilization (X 1), the weight ratio of nanoparticles-to-cell (X 2), and PEI concentration (X 3) using the response surface methodology. The best optimal setting of the variables was determined at X 1 of 6.1, X 2 of 0.6, and X 3 of 0.7 w% with the transesterification activity of 7.6 × 10 5 IU g biocatalyst − 1 gained a FAME yield of 78.4% after 3 h. The MWCBs showed an improvement in thermal stability where the optimal temperature was shifted from 34 C to 38 • C and the deactivation energy was increased from 183.9 kJ mol − 1 and 223.6 kJ mol − 1 after the immobilization. The MWCBs displayed satisfactory sustainability in the repeated transesterification experiments and maintained 87.3% of the original activity after 10 cycles which was higher than the bare-WCB system with a recovery of 57.4%.

ABSTRACT— An extracellular lipase of Aspergillus carbonarius was immobilized by adsorption onto a polyethylene terephthalate support. The optimum conditions for immobilization of lipase were studied. The optimum concentration of enzyme solution for immobilization was 35 kU/g of support, at which 2.28% immobilization efficiency was achieved. The optimum incubation time for immobilization of the enzyme was 4 hours. The optimum pH for immobilization was pH 5. The immobilized lipase retained 93% of its initial activity after 5 cycles of transesterification reaction in n-hexane medium.

1,5,6 Professor, Department of Chemical Engineering, Bharati Vidyapeeth College of Engineering, Navi Mumbai, Maharashtra, India. Abstract Conventional fuels are known for polluting air by emissions of sulfur dioxides, carbondioxides, particulate matter and other gases. Biodiesel is a renewable, biodegradable and non-toxic alternative fuel to ‘fossil’ diesel. Biodiesel manufacturers are focusing their attention on using low-cost feedstock such as waste cooking oil to ensure economic viability in biodiesel production. Waste cooking oil(WCO) is far less expensive than refined vegetable oils and therefore has become a promising alternative feedstock to produce biodiesel. WCO contain large amount of free fatty acid (FFA) and water. Enzymatic transesterification has drawn researcher's attention in last ten years due to the downstream processing problem such as huge amount of wastewater generation and difficulty in glycerol recovery posed by chemical transesterification in turn results...

1,5,6 Professor, Department of Chemical Engineering, Bharati Vidyapeeth College of Engineering, Navi Mumbai, Maharashtra, India. Abstract Conventional fuels are known for polluting air by emissions of sulfur dioxides, carbondioxides, particulate matter and other gases. Biodiesel is a renewable, biodegradable and non-toxic alternative fuel to ‘fossil’ diesel. Biodiesel manufacturers are focusing their attention on using low-cost feedstock such as waste cooking oil to ensure economic viability in biodiesel production. Waste cooking oil(WCO) is far less expensive than refined vegetable oils and therefore has become a promising alternative feedstock to produce biodiesel. WCO contain large amount of free fatty acid (FFA) and water. Enzymatic transesterification has drawn researcher's attention in last ten years due to the downstream processing problem such as huge amount of wastewater generation and difficulty in glycerol recovery posed by chemical transesterification in turn results...

District heating networks are commonly addressed in the literature as one of the most effective solutions for decreasing the greenhouse gas emissions from the building sector. These systems require high investments which are returned through the heat sales. Due to the changed climate conditions and building renovation policies, heat demand in the future could decrease, prolonging the investment return period. The main scope of this paper is to assess the feasibility of using the heat demand-outdoor temperature function for heat demand forecast. The district of Alvalade, located in Lisbon (Portugal), was used as a case study. The district is consisted of 665 buildings that vary in both construction period and typology. Three weather scenarios (low, medium, high) and three district renovation scenarios were developed (shallow, intermediate, deep). To estimate the error, obtained heat demand values were compared with results from a dynamic heat demand model, previously developed and validated by the authors. The results showed that when only weather change is considered, the margin of error could be acceptable for some applications (the error in annual demand was lower than 20% for all weather scenarios considered). However, after introducing renovation scenarios, the error value increased up to 59.5% (depending on the weather and renovation scenarios combination considered). The value of slope coefficient increased on average within the range of 3.8% up to 8% per decade, that corresponds to the decrease in the number of heating hours of 22-139h during the heating season (depending on the combination of weather and renovation scenarios considered). On the other hand, function intercept increased for 7.8-12.7% per decade (depending on the coupled scenarios). The values suggested could be used to modify the function parameters for the scenarios considered, and improve the accuracy of heat demand estimations.

The optimization method is vital in chemical synthesis and has been applied in many fields nowadays. Response surface methodology (RSM) is an example of an optimization method that is useful in examining the effects of multiple independent variables. RSM was applied in many studies to optimize the transesterification of biodiesel production from palm oil in the presence of a catalyst. This paper aims to provide an overview of recent catalyzed transesterification trends, as well as the benefits and drawbacks of heterogeneous, homogeneous, and enzyme catalysts in biodiesel production. RSM was used to design the process and statistically analyze the interaction effects of the independent reaction variables. The reaction variables, such as reaction time, reaction temperature, catalyst amount, and the molar ratio of the substrate, were optimized during the process. A statistical model and response surface plots were visualized graphically in the contour plots and three-dimensional figures to explain the interactive effects of variables on a response. In sum, this paper discussed the relationships between the reaction parameters and the production of biodiesel and the optimum conditions for biodiesel production using RSM.

The lipase from Burkholderia sp. EQ3 was used to synthesize wax esters in comparison with commercial lipases. The supernatant of Burkholderia sp. EQ3 was collected from a liquid basal medium with 1 % fish oil after 12 h cultivation (1.90 U/ml of lipase activity). The crude lipase was prepared by acetone precipitation of the culture supernatant (4.70 U/mg and 9.40 purification folds). The crude fish fat obtained by hexane extraction of waste fat from the wastewater pond of a canned tuna factory and cetyl alcohol were used for wax esters synthesis. Five commercial lipases were screened in comparison with crude lipase from Burkholderia sp. EQ3 in wax esters synthesis. The optimum conditions for wax esters synthesis from crude fish fat using Novozyme 435 were enzyme 1 U, substrate molar ratio of crude fish fat to cetyl alcohol 1:2 (115.30 mg of crude fish fat and 66.67 mg of cetyl alcohol) in hexane at 37°C and 200 rpm with 90.81 % (TLC-FID peak area) after one h of reaction. The optimum conditions for the synthesis by crude lipase from Burkholderia sp. EQ3 were crude lipase 40 U, substrate molar ratio of crude fish fat and cetyl alcohol 1:2 in isooctane at 30°C and 200 rpm with 95.07 % (TLC-FID peak area) after 6 h of reaction. The synthesized wax esters were mainly composed of cetyl palmitate and cetyl oleate. Keywords Wax esters Á Waste fat Á Fish canning industry Á Lipase Á Esterification Á Burkholderia sp.

h i g h l i g h t s Esterification of oleic acid using magnetic ionic liquid. Multi-objective optimization was performed for two responses. Both predicted yield and conversion were 83.4% at optimum conditions. Recycled ionic liquid can be used repeatedly without significant activity loss. Low activation energy and pre-exponential factor for esterification of oleic acid.

Cellulosic enzymes, including cellulase, play an important role in biotechnological processes in the fields of food, cosmetics, detergents, pulp, paper, and related industries. Low thermal and storage stability of cellulase, presence of impurities, enzyme leakage, and reusability pose great challenges in all these processes. These challenges can be overcome via enzyme immobilization methods. In recent years, cellulase immobilization onto nanomaterials became the focus of research attention owing to the surface features of these materials. However, the application of these nanomaterials is limited due to the efficacy of their recovery process. The application of magnetic nanoparticles (MNPs) was suggested as a solution to this problem since they can be easily removed from the reaction mixture by applying an external magnet. Recently, MNPs were extensively employed for enzyme immobilization owing to their low toxicity and various practical advantages. In the present review, recent adv...

Among the industrial enzymes, cellulase plays a central role in biotechnology and agricultural applications such as foods, chemicals, detergents, cosmetics, pulp, and paper. Most of these applications suffer from low thermal and storage stability of cellulase, contamination of products by enzyme and lake of enzyme reusability that make process inappropriate in economic viewpoint. For overcoming these issues and improve the stability and reusability of the enzyme, the immobilization methods could employ. Recently, the immobilization of cellulase on nanomaterials is under more considerable attention due to the significant surface properties of these supports. However, the application of these supports is also restricted by troubles in recovery, e.g., via centrifugation or filtration. This restriction could be solved using magnetic nanoparticles (MNPs) that can simply separate from the reaction media by using a simple magnet. MNPs have been commonly used for enzyme immobilization in recent years due to this practical advantage and their low toxicity. In this review, we attempt to provide a more detailed overview of the applications and recent advances of immobilization of cellulase enzyme onto different functionalized MNPs. At last, we tried to present the future perspective of this research area such as the enzyme reusability, the facile recovery way of cellulase, and the efficient biotechnology production process.

Cellulase on commercial superparamagnetic nanoparticles was characterized by DLS, and TEM methods in relation to their size and structure. The cellulase enzyme was bound via physical adsorption (ionic bound). FTIR spectroscopy confirmed the successful binding of cellulase (endoglucanase) onto the particle, and binding efficiency was determined at 95% using the Bradford method. The maximal enzyme activity was assessed using CMC as the substrate and was 0.1 unit (mol/min ml). The adsorption capacity of cellulase onto nanoparticles reached 31 mg/g. The stability of the immobilized enzyme increased in comparison with the free enzyme. Overall, this study showed that that the stability and activity of the cellulase were enhanced via physical adsorption to the magnetic nanoparticles. This suggested that immobilized enzyme on magnetic beads could be used in an interesting range of application allowing both using in broader temperature and pH ranges, facilitating long-term storage, while permitting magnetic recovery of the enzyme for reuse or purification of the product.

Magnetic nanoparticles (Fe3O4) were synthesized by co-precipitating Fe 2+ and Fe 3+ ions in an ammonia solution and treating under hydrothermal conditions. Cellulase was immobilized onto Fe3O4 magnetic nanoparticles via glutaraldehyde activation. Using response surface methodology and Box-Behnken design, the variables such as magnetic nanoparticle concentration, glutaraldehyde concentration, enzyme concentration, and cross linking time were optimized. The Box-Behnken design analysis showed a reasonable adjustment of the quadratic model with the experimental data. Statistical contour plots were generated to evaluate the changes in the response surface and to understand the relationship between the nanoparticles and the enzyme activity. Scanning electron microscopy, X-ray diffraction analysis, and Fourier transform infrared spectroscopy were studied to characterize size, structure, morphology, and binding of enzyme onto the nanoparticles. The stability and activity of the bound cellulase was analyzed using various parameters including pH, temperature, reusability, and storage stability. The immobilized cellulase was compared with free cellulase and it shows enhanced stability and activity.

Although fossil fuels have been serving mankind for several centuries, their exploration and use bring about several environmental problems. Besides, fossil fuels are neither renewable nor sustainable. There is therefore a need to search for renewable alternatives which are sustainable and environmentally friendly. Biodiesel, chemically known as methyl ester, is a renewable and sustainable biofuel used to run diesel engines and it is mainly produced by transesterification through either chemical or enzymatic catalysis. Enzyme catalyzed transesterification has several advantages over chemical catalysis. However, enzyme production and purification are expensive and the inability to recycle these enzymes has limited their application in large scale bioconversion processes. This review highlights some of the strategies employed to reduce the cost of lipase catalzsed transesterificaion, some important feed stocks used in biodiesel production, methods of processing biodiesel, and advantages of enzymatic transesterification over chemical catalyzed transesterification. The properties and sources of lipase enzymes used in enzyme catalyzed transesterfication are also highlighted and discussed. Factors that affect enzyme catalyzed transesterification are also discussed. It also highlights the possible methods of solving the problems encountered in enzymatic transesterification.

Cellulosic enzymes, including cellulase, play an important role in biotechnological processes in the fields of food, cosmetics, detergents, pulp, paper, and related industries. Low thermal and storage stability of cellulase, presence of impurities, enzyme leakage, and reusability pose great challenges in all these processes. These challenges can be overcome via enzyme immobilization methods. In recent years, cellulase immobilization onto nanomaterials became the focus of research attention owing to the surface features of these materials. However, the application of these nanomaterials is limited due to the efficacy of their recovery process. The application of magnetic nanoparticles (MNPs) was suggested as a solution to this problem since they can be easily removed from the reaction mixture by applying an external magnet. Recently, MNPs were extensively employed for enzyme immobilization owing to their low toxicity and various practical advantages. In the present review, recent adv...

Among the industrial enzymes, cellulase plays a central role in biotechnology and agricultural applications such as foods, chemicals, detergents, cosmetics, pulp, and paper. Most of these applications suffer from low thermal and storage stability of cellulase, contamination of products by enzyme and lake of enzyme reusability that make process inappropriate in economic viewpoint. For overcoming these issues and improve the stability and reusability of the enzyme, the immobilization methods could employ. Recently, the immobilization of cellulase on nanomaterials is under more considerable attention due to the significant surface properties of these supports. However, the application of these supports is also restricted by troubles in recovery, e.g., via centrifugation or filtration. This restriction could be solved using magnetic nanoparticles (MNPs) that can simply separate from the reaction media by using a simple magnet. MNPs have been commonly used for enzyme immobilization in recent years due to this practical advantage and their low toxicity. In this review, we attempt to provide a more detailed overview of the applications and recent advances of immobilization of cellulase enzyme onto different functionalized MNPs. At last, we tried to present the future perspective of this research area such as the enzyme reusability, the facile recovery way of cellulase, and the efficient biotechnology production process.

Background: Collagen, the most abundant protein in the human body, and as an extracellular matrix protein, has an important role in the fiber formation. This feature of the collagen renders establishment of the structural skeleton in tissues. Regarding specific features associated with the collagen, such as, formation of the porous structure, permeability and hydrophilicity, it can also be used as a biocompatible matrix in the enzyme engineering. Objectives: The aim of the present study was to investigate the application of the type I collagen as a matrix for alkaline phosphatase immobilization using cross-linking method. Material and Methods: The Alkaline phosphatase was covalently immobilized on collagen matrix by using 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride (EDC). The source of the alkaline phosphatase was from the bovine intestinal mucous. After that, the activity of the immobilized enzyme was assayed under different experimental conditions. Results: The optimum pH was similar to that of the free enzyme, whereas the optimum temperature and thermal stability were shown some increments. The surface topography of the collagen matrix containing immobilized enzyme and ALP (Alkaline phosphatase) deficient was investigated by Atomic-force microscopy (AFM). Images that have been obtained applying AFM show significant differences between uncovered and immobilized enzyme-matrix surface topography. Conclusions: Our findings suggest that type I collagen can be utilized as a matrix for alkaline phosphatase immobilization via cross-linking method.

In order to measure the intermolecular binding forces between two halves (or partners) of naturally split protein splicing elements called inteins, a novel thiol-hydrazide linker was designed and used to orient immobilized antibodies specific for each partner. Activation of the surfaces was achieved in one step allowing direct force measurements of the formation of a peptide bond catalyzed by the binding of the two partners of the split intein (called protein trans-splicing). Through this binding process, a whole functional intein is formed resulting in subsequent splicing. Atomic force microscopy (AFM) was used to directly measure the split intein partner binding at 1µm/s between native (wild-type) and mixed pairs of C-and N-terminal partners of naturally occurring split inteins from three cyanobacteria. Native and mixed pairs exhibit similar binding forces within the error of the measurement technique (~52 pN). Bioinformatic sequence analysis and computational structural analysis discovered a zipper-like contact between the two partners with electrostatic and non-polar attraction between multiple aligned ion pairs and hydrophobic residues. Also, we tested the Jarzynski's equality and demonstrated, as expected, that nonequilibrium dissipative measurements obtained here gave larger energies of interaction as compared with those for equilibrium. Hence, AFM coupled with our immobilization strategy and computational studies provides a useful analytical tool for the direct measurement of intermolecular association of split inteins and could be extended to any interacting protein pair.

Increasing energy demand necessitates the production of sustainable fuels, which can be in the form of bio-fuels. One of such bio-fuels is biodiesel, which is typically produced via transesterification. The development of homogeneous catalyst that is relatively easy to synthesize, cheap, reusable, and environmentally friendly, is a major issue in transesterification reaction. The use of Deep eutectic solvent (DES) as catalyst, is believed to be a significant step in the direction of attaining a sustainable bio-economy. In this study, deep eutectic solvent was synthesized from different mole ratios of K2CO3/glycerol. The synthesized DES was used as catalyst in the transesterification reaction to produce biodiesel from Jatropha curcas oil. Box-Behnken design (BBD) was used to determine the factors that significantly affect the biodiesel yield. Optimum fatty acid methyl ester (FAME) yield of 98.2845% was achieved at optimum conditions of 1:32.58 mole ratio of K2CO3/glycerol, 8.96% w/w ...

Increasing energy demand necessitates the production of sustainable fuels, which can be in the form of bio-fuels. One of such bio-fuels is biodiesel, which is typically produced via transesterification. The development of homogeneous catalyst that is relatively easy to synthesize, cheap, reusable, and environmentally friendly, is a major issue in transesterification reaction. The use of Deep eutectic solvent (DES) as catalyst, is believed to be a significant step in the direction of attaining a sustainable bio-economy. In this study, deep eutectic solvent was synthesized from different mole ratios of K 2 CO 3 /glycerol. The synthesized DES was used as catalyst in the transesterification reaction to produce biodiesel from Jatropha curcas oil. Box-Behnken design (BBD) was used to determine the factors that significantly affect the biodiesel yield. Optimum fatty acid methyl ester (FAME) yield of 98.2845% was achieved at optimum conditions of 1:32.58 mole ratio of K 2 CO 3 /glycerol, 8.96% w/w concentration of DES, and 69.58 minutes. GC-MS analysis revealed that the produced biodiesel contained 98.87% ester content. The properties of the biodiesel produced were characterized and found to agree with those of ASTM D6751-12 standard. Thus, suggesting the synthesized DES is a promising catalyst in the transesterification reaction to produce biodiesel from Jatropha curcas oil.

In this study, two highly thermotolerant and methanol-tolerant lipase-producing bacteria were isolated from cooking oil and they exhibited a high number of catalytic lipase activities recording 18.65 ± 0.68 U/mL and 13.14 ± 0.03 U/mL, respectively. Bacterial isolates were identified according to phenotypic and genotypic 16S rRNA characterization as Kocuria flava ASU5 (MT919305) and Bacillus circulans ASU11 (MT919306). Lipases produced from Kocuria flava ASU5 showed the highest methanol tolerance, recording 98.4% relative activity as well as exhibited high thermostability and alkaline stability. Under the optimum conditions obtained from 3D plots of response surface methodology design, the Kocuria flava ASU5 biocatalyst exhibited an 83.08% yield of biodiesel at optimized reaction variables of, 60 ○C, pH value 8 and 1:2 oil/alcohol molar ratios in the reaction mixture. As well as, the obtained results showed the interactions of temperature/methanol were significant effects, whereas th...

In this study, two highly thermotolerant and methanol-tolerant lipase-producing bacteria were isolated from cooking oil and they exhibited a high number of catalytic lipase activities recording 18.65 ± 0.68 U/mL and 13.14 ± 0.03 U/mL, respectively. Bacterial isolates were identified according to phenotypic and genotypic 16S rRNA characterization as Kocuria flava ASU5 (MT919305) and Bacillus circulans ASU11 (MT919306). Lipases produced from Kocuria flava ASU5 showed the highest methanol tolerance, recording 98.4% relative activity as well as exhibited high thermostability and alkaline stability. Under the optimum conditions obtained from 3D plots of response surface methodology design, the Kocuria flava ASU5 biocatalyst exhibited an 83.08% yield of biodiesel at optimized reaction variables of, 60 ○C, pH value 8 and 1:2 oil/alcohol molar ratios in the reaction mixture. As well as, the obtained results showed the interactions of temperature/methanol were significant effects, whereas th...

The chemical-physical properties of native oxide layers, spontaneously forming on crystalline Si wafers, can be strictly correlated to the dopant type and the doping level. In particular, our investigations have focused on oxide layers forming upon air exposure in a clean room after Si wafer production, for doping concentration levels from ≈ 10 13 to ≈ 10 19 cm-3. In order to determine these correlations, we have studied the surface, the bulk, and its interface with Si. The surface is investigated with contact angle, thermal desorption and atomic force microscopy obtaining information on the surface energy, cleanliness, and morphology, respectively. The thickness is measured with Ellipsometry and the chemical composition with X-ray Photoemission Spectroscopy. The electrostatic charges within the oxide layer and at the interface with Si are studied with Kelvin Probe Microscopy. We find that some properties show an abrupt change, i.e. thickness, while others present a maximum, i.e. silanol concentration and Si intermediate-oxidation states, at a critical doping concentration of ≈ 2.1•10 15 cm-3. Additionally, two electrostatic contributions originate from the silanols present on the surface and the net charge distributed within the layer. Lastly, the surface roughness also depends on the doping concentration, showing a minimum at the same critical doping concentration. These findings can be fully reproduced for oxide layers re-grown in a clean room after a chemical etching of the native ones.

Environmental concerns, along with oil shortages, have increased industrial interest in biomass conversion to produce biofuels and other valuable chemicals. A green option in biomass processing is the use of enzymes, such as cellulases, hemicellulases, and ligninolytic (laccase and peroxidases), which have outstanding specificity toward their substrates and can be reused if immobilized onto magnetic nanocarriers. Numerous studies report the biocatalysts’ performance after covalent binding or adsorption on differently functionalized magnetic nanoparticles (MNPs). Functionalization strategies of MNPs include silica-based surfaces obtained through a sol–gel process, graphene oxide-based nanocomposites, polymer-coated surfaces, grafting polymer brushes, and others, which have been emphasized in this review of the immobilization and co-immobilization of enzymes used for biomass conversion. Careful analysis of the parameters affecting the performance of enzyme immobilization for new hybri...

The aim of this work was to optimize the production of fatty acid methyl ester (FAME, biodiesel) from wet Nannchloropsis gaditana microalgal biomass by direct enzymatic transesterification. This was done in order to avoid the high cost associated with the prior steps of drying and oil extraction. Saponifiable lipids (SLs) from microalgal biomass were transformed to FAME using the lipase Novozyme 435 (N435) from Candida antarctica as the catalyst, and finally the FAME were extracted with hexane. t-Butanol was used as the reaction medium so as to decrease lipase deactivation and increase mass transfer velocity. A FAME conversion of 99.5% was achieved using wet microalgal biomass homogenized at 140 MPa to enhance cell disruption, a N435:oil mass ratio of 0.32, methanol added in 3 stages to achieve a total of 4.6 cm 3 g À1 of oil and 7.1 cm 3 g À1 oil of added t-butanol, with a reaction time of 56 h. The FAME conversion decreased to 57% after catalyzing three reactions with the same lipase batch. This work shows the influence of the polar lipids contained in the microalgal biomass both on the reaction velocity and on lipase activity.

In this study was verified the influence of different additives in the transesterification reaction of the crude coconut oil catalyzed with lipase from Burkholderia cepacia immobilized by physical adsorption onto palm fiber originated from agroindustrial wastes. Reactions was performed under the conditions: molar ratio 1:7 (oil:alcohol), 10 % biocatalyst immobilized for 96 h at 40 °C in the presence in different concentrations of additives (water, tert-butanol, molecular sieve and protic ionic liquids). The results showed that the use of all the additives in this study did not increase conversion to ethyl esters. The maximum conversion in the absence of the additives was 72 %, in the presence of water 45 ± 2 %, molecular sieve 53 ± 2 % and tert-butanol 59 ± 2 %, respectively, all in the lowest concentrations. For ionic liquids, those with higher alkyl chains had the highest conversions, but lower than in their absence.

Commercial scale microalgal biodiesel production is still challenging due to high cultivation cost and inefficient conversion technologies. This work explicated an economical cultivation strategy using anaerobic centrate as growth medium of Acutodesmus obliquus. Among various modes evaluated maximum lipid productivity of 57.03 mgL −1 d −1 was achieved under mixotrophic cultivation condition using anaerobic centrate. Conversion of microalgal lipids to biodiesel using immobilized Candida rugosa lipase was investigated. Reaction parameters viz. temperature, methanol to oil molar and enzyme amount were optimized using response surface methodology. Best conversion of 95.36% was achieved at 50°C, methanol to oil ratio of 3:1 and 15% enzyme amount based on oil weight. Immobilized C. rugosa lipase can be used for 5 batches without much loss in conversion efficiency (> 90%). Most of the fuel properties comply with ASTM and EN standards.

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A newly isolated white-rot fungus, Armillaria sp. strain F022, was isolated from the decayed wood in a tropical rain forest. Strain F022 was capable of decolorizing a variety of synthetic dyes, including azo, triphenylmethane, and anthraquinone dyes, with an optimal efficiency of decolorization obtained when dyes added after 96 h of culture, with the exception of Brilliant Green. All of the tested dyes were decolorized by the purified laccase in the absence of any redox mediators, but only a few were completely removed, while others were not completely removed even when decolorization time was increased. The laccase, with possible contributions from unknown enzymes, played a role in the decolorization process carried out by Armillaria sp. F022 cultures, and this biosorption contributed a negligible part to the decolorization by cultures. The effect of dye to fungal growth was also investigated. When dyes were added at 0 h of culture, the maximum dry mycelium weight (DMW) values in the medium containing Brilliant Green were 1/6 of that achieved by the control group. For other dyes, the DMW was similar with control. The toxic tolerance of dye for the cell beads was excellent at least up to a concentration of 500 mg/l. The optimum conditions for decolorization of three synthetic dyes are at pH 4 and 40°C.

Increasing energy demand necessitates the production of sustainable fuels, which can be in the form of bio-fuels. One of such bio-fuels is biodiesel, which is typically produced via transesterification. The development of homogeneous catalyst that is relatively easy to synthesize, cheap, reusable, and environmentally friendly, is a major issue in transesterification reaction. The use of Deep eutectic solvent (DES) as catalyst, is believed to be a significant step in the direction of attaining a sustainable bio-economy. In this study, deep eutectic solvent was synthesized from different mole ratios of K2CO3/glycerol. The synthesized DES was used as catalyst in the transesterification reaction to produce biodiesel from Jatropha curcas oil. Box-Behnken design (BBD) was used to determine the factors that significantly affect the biodiesel yield. Optimum fatty acid methyl ester (FAME) yield of 98.2845% was achieved at optimum conditions of 1:32.58 mole ratio of K2CO3/glycerol, 8.96% w/w ...

In this study was verified the influence of different additives in the transesterification reaction of the crude coconut oil catalyzed with lipase from Burkholderia cepacia immobilized by physical adsorption onto palm fiber originated from agroindustrial wastes. Reactions was performed under the conditions: molar ratio 1:7 (oil:alcohol), 10 % biocatalyst immobilized for 96 h at 40 °C in the presence in different concentrations of additives (water, tert-butanol, molecular sieve and protic ionic liquids). The results showed that the use of all the additives in this study did not increase conversion to ethyl esters. The maximum conversion in the absence of the additives was 72 %, in the presence of water 45 ± 2 %, molecular sieve 53 ± 2 % and tert-butanol 59 ± 2 %, respectively, all in the lowest concentrations. For ionic liquids, those with higher alkyl chains had the highest conversions, but lower than in their absence.

1,5,6 Professor, Department of Chemical Engineering, Bharati Vidyapeeth College of Engineering, Navi Mumbai, Maharashtra, India. Abstract Conventional fuels are known for polluting air by emissions of sulfur dioxides, carbondioxides, particulate matter and other gases. Biodiesel is a renewable, biodegradable and non-toxic alternative fuel to ‘fossil’ diesel. Biodiesel manufacturers are focusing their attention on using low-cost feedstock such as waste cooking oil to ensure economic viability in biodiesel production. Waste cooking oil(WCO) is far less expensive than refined vegetable oils and therefore has become a promising alternative feedstock to produce biodiesel. WCO contain large amount of free fatty acid (FFA) and water. Enzymatic transesterification has drawn researcher's attention in last ten years due to the downstream processing problem such as huge amount of wastewater generation and difficulty in glycerol recovery posed by chemical transesterification in turn results...

Biodiesel, fatty acid methyl ester produced by the transesterification of vegetable oil with methanol, is a promising alternative to petroleum-based diesel fuel. In the present study, the methanolysis of high free fatty acid (FFA) Jatropha curcas oil in a transesterification reaction using K 2 CO 3 /CaO solid base catalyst was studied. The various reaction parameters in the transesterification reaction were also discussed. The catalyst was characterized by means of Fourier transform infrared, X-ray diffraction, temperature programmed desorption of CO 2 , scanning electron microscopy, particle size analyzer, true density, and surface area analyzer. The optimum conversion of jatropha oil was 92% when the transesterification reaction was carried out at 70 • C with 10:1 molar ratio of methanol to oil at reaction time of 3 h and catalyst amount of 6 wt%. The efficiency of catalysts in the methanolysis of jatropha oil was also investigated.

The aim of this study was to produce epoxidized monoalkyl esters (EMAE), a valuable class of oleochemicals used in a wide range of products and industries, from used soybean cooking oil (USCO) and fusel oil via a three-step chemoenzymatic process. This process consists of a first enzymatic hydrolysis of USCO to produce free fatty acids (FFA). Here, five microbial lipases with different specificities were tested as biocatalysts. Full hydrolysis of USCO was obtained after a 180 min reaction time under vigorous stirring (1500 rpm) using a non-specific lipase from Candida rugosa (CRL). Then, monoalkyl esters (MAE) were produced via the esterification of FFA and fusel oil in a solvent-free system using the lipase Eversa® Transform 2.0 (ET2.0) immobilized via physical adsorption on poly(styrenene-divinylbenzene) (PSty-DVB) beads as a biocatalyst. Different water removal strategies (closed and open reactors in the presence or absence of molecular sieves at 5% m.m−1) on the reaction were ev...

The lipase from Burkholderia sp. EQ3 was used to synthesize wax esters in comparison with commercial lipases. The supernatant of Burkholderia sp. EQ3 was collected from a liquid basal medium with 1 % fish oil after 12 h cultivation (1.90 U/ml of lipase activity). The crude lipase was prepared by acetone precipitation of the culture supernatant (4.70 U/mg and 9.40 purification folds). The crude fish fat obtained by hexane extraction of waste fat from the wastewater pond of a canned tuna factory and cetyl alcohol were used for wax esters synthesis. Five commercial lipases were screened in comparison with crude lipase from Burkholderia sp. EQ3 in wax esters synthesis. The optimum conditions for wax esters synthesis from crude fish fat using Novozyme 435 were enzyme 1 U, substrate molar ratio of crude fish fat to cetyl alcohol 1:2 (115.30 mg of crude fish fat and 66.67 mg of cetyl alcohol) in hexane at 37°C and 200 rpm with 90.81 % (TLC-FID peak area) after one h of reaction. The optimum conditions for the synthesis by crude lipase from Burkholderia sp. EQ3 were crude lipase 40 U, substrate molar ratio of crude fish fat and cetyl alcohol 1:2 in isooctane at 30°C and 200 rpm with 95.07 % (TLC-FID peak area) after 6 h of reaction. The synthesized wax esters were mainly composed of cetyl palmitate and cetyl oleate. Keywords Wax esters Á Waste fat Á Fish canning industry Á Lipase Á Esterification Á Burkholderia sp.

In this study was verified the influence of different additives in the transesterification reaction of the crude coconut oil catalyzed with lipase from Burkholderia cepacia immobilized by physical adsorption onto palm fiber originated from agroindustrial wastes. Reactions was performed under the conditions: molar ratio 1:7 (oil:alcohol), 10 % biocatalyst immobilized for 96 h at 40 °C in the presence in different concentrations of additives (water, tert-butanol, molecular sieve and protic ionic liquids). The results showed that the use of all the additives in this study did not increase conversion to ethyl esters. The maximum conversion in the absence of the additives was 72 %, in the presence of water 45 ± 2 %, molecular sieve 53 ± 2 % and tert-butanol 59 ± 2 %, respectively, all in the lowest concentrations. For ionic liquids, those with higher alkyl chains had the highest conversions, but lower than in their absence.

Clean renewable energy is one of the burning problems of this century. Fossil fuels, global warming, carbon emissions threaten the survival and expansion of the human race as a whole. In this critical juncture, it is imperative that we shift to renewable, cleaner fuels and reduce our dependence on the conventional fossil fuels we’ve been accustomed to use. Lipase is an enzyme which catalyzes the hydrolysis of lipids resulting in the formation of saturated as well as unsaturated fatty acid chains and glycerol. The saturated/unsaturated fatty acid chains being highly reduced releases a huge amount of energy on complete oxidation. This not only acts as a clean viable substitute to the fossil fuels we use but also reduces the pollution caused by the disposal of used waste vegetable oils which aren’t fit for human consumption. Lipase, being an enzyme can be reused and this technique is much more efficient and less polluting than the conventional base/acid catalyzed transesterification co...

Increasing energy demand necessitates the production of sustainable fuels, which can be in the form of bio-fuels. One of such bio-fuels is biodiesel, which is typically produced via transesterification. The development of homogeneous catalyst that is relatively easy to synthesize, cheap, reusable, and environmentally friendly, is a major issue in transesterification reaction. The use of Deep eutectic solvent (DES) as catalyst, is believed to be a significant step in the direction of attaining a sustainable bio-economy. In this study, deep eutectic solvent was synthesized from different mole ratios of K2CO3/glycerol. The synthesized DES was used as catalyst in the transesterification reaction to produce biodiesel from Jatropha curcas oil. Box-Behnken design (BBD) was used to determine the factors that significantly affect the biodiesel yield. Optimum fatty acid methyl ester (FAME) yield of 98.2845% was achieved at optimum conditions of 1:32.58 mole ratio of K2CO3/glycerol, 8.96% w/w ...

Biodiesel produced from microalgae biomass has been pursued as a possible replacement to petroleum diesel. Among the main steps in microalgae biodiesel production are the drying and cell walls disruption, which are energy intensive and/or time consuming, and oil extraction, which is conventionally done using toxic organic solvents that contaminate the left over biomass and require additional solvent recovery. Therefore, these steps are considered the major obstacles facing the commercialization of microalgae biodiesel. xii

The world of biosensors is expanding at a rapid pace with an ever-increasing demand for more sensitive miniaturized devices. Acoustic wave biosensors are not spared from this trend. In this domain, the search for enhanced sensitivity is increasingly oriented toward the rational design of new piezoelectric materials with superior properties to substitute for prevalent quartz. With respect to surface chemistry, construction of the biorecognition element, more often than not, requires the use of bifunctional molecules that can spontaneously assemble on the substrate and form organic surfaces readily biofunctionalizable in a subsequent, ideally single step. In this context, we present herein the surface modification of aluminum nitride (AlN) with alkyltrichlorosilane cross-linking molecules bearing a functionalizable benzenethiosulfonate moiety. This latter feature is next demonstrated through the straightforward, preactivation-free immobilization of thiolated biotin probes. To date, AlN has only received little attention in the field of piezoelectric biosensors despite its many attractive properties and the perspective to operate devices at ultra-high frequencies (GHz) with unprecedented sensitivity. To our knowledge, this work describes one of the first examples of direct surface derivatization of AlN with bifunctional trichlorosilane molecules. It also constitutes a first step toward the development of electrodeless GHz piezoelectric biosensing platforms based on AlN and trichlorosilane surface chemistry.