Activity Of Free Enzyme Research Articles

Seed fortification with chicory (<italic>Cichorium intybus</italic> L.) leaf extract to enhance seed vigour, viability and productivity in blackgram (<italic>Vigna mungo</italic> L.)

Study was carried out to evaluate the effect of herbal fortification of seeds with chicory leaf extract on seed physiological properties related to vigour, viability and yield potential in blackgram cv. ADT 3. The treatment was effected through soaking of one month old seeds with fresh chicory (<italic>Cichorium intybus</italic> L) leaf extract at 10, 20 and 25 per cent concentrations followed by dehydration in a drying chamber at 30° C for three days to reach original moisture content (10 ± 0.25% on wet weight basis). Part of the treated seeds was subjected to artificial and natural ageing process. Treated and aged seeds when evaluated through germination per cent, speed of germination, dry matter production and biochemical properties, significant differences were evident with 20 per cent concentration. The magnitude of reduction after ageing in respect of the aforesaid parameters was more in control and the least in herbal fortified seeds. Treated seeds (20% extract) exhibited higher activity of free radical scavenging enzyme of catalase (2122 units), lower amounts of leachate amino acids (40.15 μg) and lipid peroxidation value (0.144) and also an enhancement in dry weight accumulation (20.23 g), leaf area index (5.35) and net assimilation rate (0.41 mg) leading to higher yield per plant (6.15 g) under field conditions.

Novel biohybrids of layered double hydroxide and lactate dehydrogenase enzyme: Synthesis, characterization and catalytic activity studies

The present work introduces new biohybrid materials involving layered double hydroxides (LDH) and biomolecule such as enzyme to produce bioinorganic system. Lactate dehydrogenase (Lac Deh) has been chosen as a model enzyme, being immobilized onto MgAl and ZnAl LDH materials via direct ion-exchange (adsorption) and co-precipitation methods. The immobilization efficiency was largely dependent upon the immobilization methods. A comparative study shows that the co-precipitation method favors the immobilization of great and tunable amount of enzyme. The structural behavior, chemical bonding composition and morphology of the resulting biohybrids were determined by X-ray diffraction (XRD) study, Fourier transform infrared (FTIR) spectroscopy and transmission electron microscopy (TEM), respectively. The free and immobilized enzyme activity and kinetic parameters were also reported using UV–Visible spectroscopy. However, the modified LDH materials showed a decrease in crystallinity as compared to the unmodified LDH. The change in activity of the immobilized lactate dehydrogenase was considered to be due, to the reduced accessibility of substrate molecules to the active sites of the enzyme and the partial conformational change of the Lac Deh molecules as a result of the immobilization way. Finally, it was proven that there is a correlation between structure/microstructure and enzyme activity dependent on the immobilization process.

A biosensor based on Coriolopsis gallica laccase immobilized on nitrogen-doped multiwalled carbon nanotubes and graphene oxide for polyphenol detection

The use of nanomaterials allows the design of ultrasensitive biosensors with advantages in the detection of organic molecules. Catechol and catechin are molecules that occur naturally in fruits, and their presence in products like dyes and wines affects quality standards. In this study, catechol and catechin were measured at the nanoscale by means of cyclic voltammetry. The oxidation of Coriolopsis gallica laccase immobilized on nitrogen-doped multiwalled carbon nanotubes (Lac/CNx-MWCNT) and on graphene oxide (Lac/GO) was used to measure the concentrations of catechol and catechin. Nitrogen-doped multiwalled carbon nanotubes (CNx-MWCNT) were synthesized by spray pyrolysis and characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and x-ray photoelectron spectroscopy (XPS). Covalently bonded hybrids with laccase (Lac/CNx-MWCNT and Lac/GO) were generated. Catalytic activity of free enzymes determined with syringaldazine yielded 14 584 UmL−1. With Lac/CNx-MWCNT at concentrations of 6.4 mmol L−1 activity was 9326 U mL−1, while enzyme activity measured with Lac/GO at concentration of 6.4 mmol L−1 was 9 234 U mL−1. The Lac/CNx-MWCNT hybrid showed higher stability than Lac/GO at different ethyl alcohol concentrations. The Lac/CNx-MWCNT hybrid can measure concentrations, not previously reported, as low as 1 × 10−8 mol L−1 by measuring the electric current responses.

Effect of metal contamination on microbial enzymatic activity in soil

Anthropogenic metal contamination is a pervasive problem in many urban or industrial areas. The interaction of metals with native soil communities is an important area of research as scientists strive to understand effects of long-term metal contamination on soil properties. Measurements of free soil enzyme activities can serve as useful indicators of microbial metabolic potential. The goals of this study are to determine extracellular soil enzymatic activities with respect to corresponding metal concentrations within a site of long-term contamination. These data are examined to understand relationships between extracellular soil enzyme activities and persistent metal loads in situ. Here we present such results from a rare research opportunity at an un-remediated, urban brownfield in Jersey City, NJ, USA. The soils of the site developed over the last 150 years through the dumping of urban fill from New York City as well as industrial rail use. The site was abandoned and fenced in the late 1960s, and within it, there is a mapped gradient of metal concentration in the soils, including As, Pb, Cr, Cu, Zn, and V. We measured soil enzymatic potential (alkaline phosphatase, cellobiohydrolase, and l-leucine-amino-peptidase) across four plots within the site and at an uncontaminated reference site that is of the same successional age and geographic influence. We found the highest enzymatic activities for all three activities measured at the site with the greatest soil metal loads and a particularly strong relationship among enzyme activity and the metals V and Cr. Our results differ from many experimental studies that show decreased soil enzyme activity in soils experimentally treated with metals. The results may indicate the effects of long-term adaptation of soil communities within these metal contaminated soils.

Bioconjugation of gold-polymer core–shell nanoparticles with bovine serum amine oxidase for biomedical applications

Core–shell gold nanoparticles [AuNPs], stabilized with a hydrophilic polymer, poly(3-dimethylammonium-1-propyne hydrochloride) [PDMPAHCl], have been used for the immobilization of bovine serum amine oxidase [BSAO]. The functionalized surface of the hybrid nanoparticles is pH responsive, due to the presence of aminic groups that carry out a double role: on one hand they act as ligands for the gold nanoparticle surface, allowing the colloidal stabilization and, on the other hand, they give a hydrophilic characteristic to the whole colloidal suspension. The core–shell nanoparticles [Au@PDMPAHCl] have been characterized by using UV–vis and X-ray photoelectron spectroscopy, DLS, ζ-potential measurements and by FE-TEM microscopy. BSAO enzyme can be loaded by non-covalent immobilization onto Au@PDMPAHCl nanoparticles up to 70% in weight, depending on the pH values of the environmental medium. Activity tests on Au@PDMPAHCl-BSAO bioconjugates confirm an enzymatic activity up to 40%, with respect to the free enzyme activity. Moreover, our results show that loading and enzymatic activity are rather interrelated characteristics and that, under appropriate polymer concentration and pH conditions, a satisfactory compromise can be reached. These results, as a whole, indicate that Au@PDMPAHCl-BSAO bioconjugate systems are promising for future biomedical applications.

Impact of Solvent pH on Direct Immobilization of Lysosome-Related Cell Organelle Extracts on TiO₂ for Melanin Treatment.

Techniques for immobilizing effective enzymes on nanoparticles for stabilization of the activity of free enzymes have been developing as a pharmaceutical field. In this study, we examined the effect of three different pH conditions of phosphate buffer, as a dissolving solvent for lysosomal enzymes, on the direct immobilization of lysosomal enzymes extracted from Hen's egg white and Saccharomyces cerevisiae. Titanium(IV) oxide (TiO2) nanoparticles, which are extensively used in many research fields, were used in this study. The lysosomal enzymes immobilized on TiO2 under each pH condition were evaluated to maintain the specific activity of lysosomal enzymes, so that we can determine the degree of melanin treatment in lysosomal enzymes immobilized on TiO2. We found that the immobilization efficiency and melanin treatment activity in both lysosomal enzymes extracted from Hen's egg white and S. cerevisiae were the highest in an acidic condition of phosphate buffer (pH 4). However, the immobilization efficiency and melanin treatment activity were inversely proportional to the increase in pH under alkaline conditions. In addition, enhanced immobilization efficiency was shown in TiO2 pretreated with a divalent, positively charged ion, Ca(2+), and the melanin treatment activity of immobilized lysosomal enzymes on TiO2 pretreated with Ca(2+) was also increased. Therefore, this result suggests that the immobilization efficiency and melanin treatment activity of lysosomal enzymes can be enhanced according to the pH conditions of the dissolving solvent.

Hydrolytic effects of scaffolding proteins CbpB and CbpC on crystalline cellulose mediated by the major cellulolytic complex from Clostridium cellulovorans

The role of the scaffolding proteins, cellulose binding protein B and C (CbpB and CbpC, respectively) were identified in cellulolytic complex (cellulosome) of Clostridium cellulovorans for efficient degradation of cellulose. Recombinant CbpB and CbpC directly anchored to the cell surface of C. cellulovorans. In addition, CbpB and CbpC showed increased hydrolytic activity on crystalline cellulose incubated with exoglucanase S (ExgS) and endoglucanase Z (EngZ) compared with the activity of free enzymes. Moreover, the results showed synergistic effects of crystalline cellulose hydrolytic activity (1.8- to 2.2-fold) when CbpB and CbpC complex with ExgS and EngZ are incubated with cellulolytic complex containing mini-CbpA. The results suggest C. cellulovorans critically uses CbpB and CbpC, which can directly anchor cells for the hydrolysis of cellulosic material with the major cellulosome complex.

Effect of excess dietary L-valine on laying hen performance, egg quality, serum free amino acids, immune function and antioxidant enzyme activity

The aim of this study was to evaluate the tolerance of laying hens for an excessive L-valine (L-val) supply on laying performance, egg quality, serum free amino acids, immune function and antioxidant enzyme activities of laying hens.A total of 720 HyLine Brown hens were allocated to 5 dietary treatment groups, each of which included 6 replicates of 24 hens, from 40 to 47 weeks of age. Graded amounts of L-val were added to the basal diet to achieve concentrations of 0 (control), 1, 2, 3 and 4 g/kg, respectively, in the experimental diets.Supplementing the diet with L-val did not affect egg production, egg mass, egg weight, feed conversion ratio (FCR) or egg quality. The average daily feed intake response to supplemental L-val was quadratic and was maximised at 2.0 g L-val/kg diet. No differences were observed for total protein, total amino acids, blood urea nitrogen (BUN), uric acid, lactate dehydrogenase (LDH), alkaline phosphatase (AKP), Ca and P concentrations among the treatments.Serum albumin concentration increased significantly in response to supplemental L-val and was also maximised at 2.0 g/kg. In addition, serum glucose increased quadratically to peak at 2.0 g L-val/kg diet. Serum free valine increased as L-val concentration increased to 2.0 g/kg diet and then decreased linearly.Supplementation of L-val did not affect the serum concentrations of total antioxidative capability (T-AOC), superoxide dismutase (SOD) and malondialdehyde (MDA). L-val supplementation did not affect the concentrations of immunoglobulins IgG, IgA, IgM and complements (C3 and C4). Serum concentration of triiodothyronine (T3) increased significantly at 2.0 g L-val/kg diet.It is concluded that high concentrations of L-val are tolerated and can be successfully supplemented into diets without detrimental effects on laying performance or immune function of laying hens.

Shape-controlled synthesis of Fe3O4/CeO2hybrid octahedra for lipase immobilization

Fe3O4/CeO2 hybrid octahedra have been successfully synthesized on the large scale via a solvothermal route, which were confirmed by a series of modern analytical tools. The formation and evolution of Fe3O4/CeO2 hybrid octahedra were systematically investigated, and a kinetically induced anisotropic growth mechanism clearly contributes to the formation of the novel architecture. Because of the magnetic nature and unique surfaces, the as-prepared Fe3O4/CeO2 hybrid octahedra were employed as magnetic carriers for lipase immobilization. Our results indicated that the lipase loading amount on the magnetic Fe3O4/CeO2 nanoparticles was about 43.1 mg of protein per g. Hydrolytic activity tests revealed that the immobilized lipase retains about 92.4% of the free enzyme's activity. The reusability assay revealed that the immobilized lipase can be reused 3 times without marked loss of catalytic activity (<5%). Most importantly, the thermal, acid and alkali stabilities of immobilized lipase were strikingly improved. It is hoped that the Fe3O4/CeO2 hybrid octahedra may find applications in biotechnology and biocatalysis.

Physicochemical changes and enzymatic activity of pre-harvest sprouting wheat

O trigo na colheita está exposto a danos por chuva, que podem levar à alteração na qualidade e perda de valor comercial. O objetivo desse trabalho foi caracterizar as alterações de diferentes variedades de trigo da região Sul que sofreram danos por chuva, detectado pelo método de falling number. Treze amostras foram avaliadas quanto a peso hectolitro, peso de mil sementes, proteína, açúcares redutores, nitrogênio solúvel, nitrogênio de aminoácidos livres e atividade enzimática (α e β-amilase, β-glucanase e protease). As alterações na fração de gliadina foram avaliadas por eletroforese SDS-PAGE. As variáveis açúcares redutores, nitrogênio solúvel, nitrogênio de aminoácidos livres, atividade de α-amilase, β-glucanase e protease apresentaram correlações significativas com falling number. À medida que houve redução do falling number, as gliadinas apresentaram alterações nas bandas entre 40 e 30 KDa que ficaram mais intensas.

Antioxidant Capacities of Flavones and Benefits in Oxidative-Stress Related Diseases

Flavonoids, a group of secondary metabolites widely distributed in the plant kingdom, have been acknowledged for their interesting medicinal properties. Among them, natural flavones, as well as some of their synthetic derivatives, have been shown to exhibit several biological activities, including antioxidant, anti-inflammatory, antitumor, anti-allergic, neuroprotective, cardioprotective and antimicrobial. The antioxidant properties of flavones allow them to demonstrate potential application as preventive and attenuating agents in oxidative stress, i.e., a biological condition that is closely associated to aging process and several diseases. Some flavones interfere in distinct oxidative-stress related events by directly reducing the levels of intracellular free radicals (hydroxyl, superoxide and nitric oxide) and/or of reactive species (e.g. hydrogen peroxide, peroxynitrite and hypochlorous acid) thus preventing their amplification and the consequent damage of other biomolecules such as lipids, proteins and DNA. Flavones can also hinder the activity of central free radical-producing enzymes, such as xanthine oxidase and nicotinamide adenine dinucleotide phosphate oxidase (NADPH-oxidase) or inducible nitric oxide synthase (iNOS) and can even modulate the intracellular levels of pro-oxidant and/or antioxidant enzymes. The evaluation of flavones antioxidant ability has been extensively determined in chemical or biological in vitro models, but in vivo therapy with individual flavones or with flavones-enriched extracts has also been reported. The present manuscript revises relevant studies focusing the preventive effects of flavones on stress-related diseases, namely the neurological and cardiovascular diseases, and diabetes and its associated complications.

Effects of porogen and cross-linking agents on improved properties of silica-supported macroporous chitosan membranes for enzyme immobilization.

A series of silica-supported macroporous chitosan membranes (CM15, CM20, and CM25) was prepared by varying the ratio of 70-230-μm-sized silica particles. These synthesized membranes were further cross-linked using different cross-linking agents for covalent immobilization of biological macromolecules especially enzymes and in this study, Bovine serum albumin and laccase. Effects of silica particle and cross-linking agents on their flow rates, surface properties, and chemical and biological properties were explored. Pore size of as-synthesized membranes was 0.1192, 0.1268, and 0.1623 μm, respectively, for CM15, CM20, and CM25. The effect of various parameters such as temperature and pH on the relative activity of both free and immobilized enzymes was studied in details. The relative enzyme activity upon immobilization was greatly enhanced several folds of its original activity. The stability of enzymes over a range of temperature and pH was significantly improved by immobilization. The optimum temperature and pH were determined to be 50 °C and pH 3, respectively, for both the free and the immobilized enzymes. The immobilized enzyme possessed good operational stability and reusability properties that support its potentiality for practical applications. Among three membranes, CM25 is confirmed to be efficient candidate due to its improved characteristics.

Immobilization of Aspergillus oryzae β-Galactosidase on Cellulose Acetate-Polymethylmethacrylate Membrane and Its Application in Hydrolysis of Lactose from Milk and Whey.

The present study demonstrates the immobilization of Aspergillus oryzae β-galactosidase on cellulose acetate-polymethylmethacrylate (CA-PMMA) membrane and its application in hydrolyzing lactose in dairy industries. The effect of physical and chemical denaturants like pH, temperature, product inhibition by galactose, storage stability, and reuse number of the enzyme immobilized on CA-PMMA membrane has been investigated. Lactose was hydrolyzed from milk and whey in batch reactors at 50°C by free and immobilized β-galactosidase (IβG). Optimum pH for the free and immobilized enzyme was found to be the same, that is, 4.5. However, IβG retained greater fractions of catalytic activity at lower and higher pH ranges. The temperature optimum for the immobilized enzyme was increased by 10°C. Moreover, Michaelis-Menten constant was increased for IβG as compared to the native one while maximum reaction rate was reduced for the immobilized enzyme. The preserved activity of free and immobilized enzyme was found to be 45% and 83%, respectively, after five weeks of storage at 4°C. Reusability of IβG was observed to be 86% even after fifth repeated use, thereby signifying its application in lactose hydrolysis (as shown in lab-scale batch reactors) in various dairy products including milk and whey.

Horseradish peroxidase encapsulation in alginate microspheres in the presence of imidazolium ionic liquids

Biocatalysis with free enzymes may not be economically viable due to the complexity of their recovery in the reaction medium. With the objective of enabling the total activity recovery yield of biocatalyst and to improve the operational characteristics, have been applied enzyme immobilization techniques. The main interest in the enzyme immobilization is to obtain a catalyst whose activity and stability is not affected during the process when compared to the free enzyme. The objective of this work was to immobilize horseradish peroxidase (HRP) by encapsulation method in alginate microspheres in the presence of imidazolium ionic liquid (ILs). The enzyme encapsulation was carried with the enzyme incorporation in an aqueous solution of sodium alginate to 0.05 % (w / v). The influence of the enzyme loading was studied in the range of 0.0406-0.65 mg HRP / g alginate. Alginate microspheres were obtained by dripping in calcium chloride solution (1M) according to literature [1]. The HRP immobilization in the presence of ILs was conducted using the enzyme loading more appropriate, incorporating in the immobilization medium 1% (w/v) of IL. The ILs studied as additives were: [C4mim]TF2N; [C4mim]BF4; [C4mim]HSO4; [C4mim]Ac e [C4mim]PF6. The activity of free and immobilized enzyme was verified by colorimetric method based on the change of absorbance at 470 nm due to the formation of product in guaiacol oxidation for three minutes. The results obtained for biocatalysts immobilized by encapsulation suggest that the use of IL as an additive in the immobilization process causes a significant increase in the total activity recovery yield (RA) HRP (increase from 5.2 to 23.5 %). The results for the HRP immobilization showed that RA was 23.5% and 20.40% for the LIs [C4mim] HSO4 and [C4mim]TF2N, respectively. [C4mim]Ac provided RA of 13.3 %, while the biocatalyst with [C4mim] PF6 showed RA of 17.3 %. The [C4mim] BF4 showed the lowest performance as an additive among the ILs studied. According to Diego et al.(2009) [2] there is not have a rule to predict the behavior of immobilized enzymes in the presence of ILs. The results obtained for biocatalysts immobilized by encapsulation in alginate microspheres show that the use of ionic liquids in this process had a positive effect on the activity of the immobilized biocatalyst. Additional studies in the structural characteristic of the immobilized biocatalyst are being conducted to evaluate the influence of ILs in the supporting structure.

Laccase immobilization on radiation synthesized epoxy functionalized polyethersulfone beads and their application for degradation of acid dye

The enzymatic degradation of textile dyes offers an alternative approach over the conventional waste water treatment processes. Covalent immobilization of the enzymes onto insoluble supports not only permits their reusability and easy separation of enzymes from the product but also, if properly performed, may improve their activities, stability and hence reduces the cost of process. In the present work, epoxy functionalized polyethersulfone (PES) was synthesized via radiation induced polymerization of poly(2,3-epoxypropyl methacrylate) [poly(EPMA)] in PES-NMP (N-methyl-2-pyrrolidone) solution using 60Co-gamma radiation source. Poly(EPMA)-functionalized-PES [poly(EPMA)-f-PES] beads were fabricated via phase inversion route using water as the anti solvent. Laccase was covalently immobilized on to the beads via one step-room temperature coupling reaction of amine group of enzyme with the epoxy group of poly(EPMA). Enzyme activity was assayed using 2,2′-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS) as the substrate. Effect of temperature and pH on the free and immobilized enzyme activity was studied. The immobilized laccase was successfully employed to degrade Acid Red 1 (AR1) dye in aqueous solution. Room temperature incubation of the laccase immobilized poly(EPMA)-f-PES beads with AR1 dye (∼10 ppm) resulted in ∼88% degradation of the dye over a period of 15 days.

Synthesis of amine-functionalized Fe3O4@C nanoparticles for lipase immobilization

Amine-functionalized Fe3O4@C nanoparticles have been successfully synthesized on a large scale via a solvothermal route. The morphology, structure and properties of the Fe3O4@C nanoparticles were investigated through different analytical tools. Due to the magnetic nature and the presence of amine-functionalized groups, the as-prepared Fe3O4@C nanoparticles were employed as magnetic carriers for lipase immobilization. Our results indicate that the lipase loading amount on the magnetic Fe3O4@C nanoparticles was about 115.6 mg of protein per g. The factors related to the catalytic activity of the immobilized lipase on the magnetic carriers were systematically investigated. Hydrolytic activity tests reveal that the immobilized lipase retains about 93% of the free enzyme activity. It should be mentioned that the thermal stability of the immobilized lipase was strikingly higher than that of the free lipase. Most importantly, the immobilized lipase retained more than 68% of its initial activities after 10 times reuse. It is hoped that the amine-functionalized Fe3O4@C nanoparticles may find an application in biotechnology and catalysis.

Preparation of Dopamine/Titania Hybrid Nanoparticles through Biomimetic Mineralization and Titanium(IV)–Catecholate Coordination for Enzyme Immobilization

In this study, a facile approach is proposed to prepare dopamine/titania hybrid nanoparticles (DTHNPs), which are synthesized via directly blending titanium(IV) bis(ammonium lactato) dihydroxide (Ti-BALDH) and dopamine aqueous solution. The amino group in dopamine is mainly in charge of inducing the hydrolysis and condensation of titanium precursor to form titania, and the catechol group in dopamine acts as an organic ligand to form titanium(IV)–catecholate coordination. These DTHNPs were characterized by tranmission electron miscroscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). The morphology of DTHNPs is changed from slightly cotton-shaped aggregates to monodisperse nanoparticles with the increase of dopamine concentration. As a model enzyme, catalase (CAT) is entrapped in the DTHNPs during the nanoparticle preparation process. Surprisingly, the entrapment efficiency of CAT can be high up to nearly 100%, and no enzyme leakage could be detected. Moreover, immobilized CAT possesses 90% the catalytic activity of free enzyme.

A comparative study of the effectiveness of β-glucosidase immobilized on CNT-nanoparticles and Ca-alginate beads

Enzymes are extensively used in various industrial, biomedical and biopharmaceutical applications. However, enzymes in their free form are unstable and expensive besides being characteristically susceptible to inhibition by high product concentrations and are highly sensitive to pH and temperature changes. Immobilization technology offers solutions to these challenges besides enhancing operational stability, longevity and ease of separation. β-glucosidase has been widely employed as model enzyme for enzymatic studies. Ca-alginate beads provide a gentle environment for immobilization, but have certain limitations such as low stability, high porosity and limitations in biocompatibility. Carbon nanotubes (CNTs) on the other hand have excellent mechanical, thermal and electrical properties, as well as dimensional and chemical compatibility with biomolecules like DNA and enzymes, suitable for biosensor design. Here, β-glucosidase was immobilized in Ca-alginate gel and multi-walled carbon nanotubes (MWCNT) using standard techniques and their activity was compared with that of free enzyme. The activity was found highest (12.53 U/mL) for the free enzyme and lowest (9.768 U/mL) for the immobilized Ca-alginate. The activity of immobilized MWCNT (12.20 U/mL) was close to the free enzyme activity. The enzyme reaction was found to follow Michaelis-Menten kinetics. The Michaelis constants, Km and Vmax, determined using Langmuir linearized plot are respectively 0.09048 μmol/mL and 0.00989 μmol/mL.min for immobilized Ca-alginate; and 0.0985 μmol/mL and 0.01237 μmol/mL.min for immobilized MWCNT. The corresponding values for the free enzyme are 0.0854 μmol/mL and 0.01263 μmol/mL.min. Thus, MWCNT appears to be a promising support material for enzyme immobilization. Keywords: β-glucosidase, immobilization, Ca-alginate, carbon-nanotube, Michaelis-Menten kinetics

Microencapsulation of β-galactosidase with different biopolymers by a spray-drying process

The aim of this work was to investigate the possibility of producing microparticles containing β-galactosidase, using different biopolymers (arabic gum, chitosan, modified chitosan, calcium alginate and sodium alginate) as encapsulating agents by a spray-drying process. This study focused on the enzyme β-galactosidase, due to its importance in health and in food processing. Encapsulation of β-galactosidase can increase the applicability of this enzyme in different processes and applications. A series of β-galactosidase microparticles were prepared, and their physicochemical structures were analyzed by laser granulometry analysis, zeta potential analysis, and by scanning electron microscopy (SEM). Microparticles with a mean diameter around 3μm have been observed, for all the biopolymers tested. The microparticles formed with chitosan or arabic gum presented a very rough surface; on the other hand, the particles formed with calcium or sodium alginate or modified chitosan presented a very smooth surface. The activity of the enzyme was studied by spectrophotometric methods using the substrate ONPG (O-nitrophenyl-β,d-galactopyranoside). The microencapsulated β-galactosidase activity decreases with all the biopolymers. The relative enzyme activity is 37, 20, 20 and 13%, for arabic gum, modified chitosan, calcium alginate and sodium alginate, respectively, when compared with the free enzyme activity. The enzyme microparticles formed with arabic gum shows the smallest decrease of Vmax, followed by the calcium alginate, sodium alginate, and modified chitosan.

Production of an endoinulinase from Aspergillus niger AUMC 9375, by solid state fermentation of agricultural wastes, with purification and characterization of the free and immobilized enzyme.

Two different substrates, sunflower (Helianthus annuus L.) tubers and lettuce (Lactuca sativa) roots, were tested. Using a mixture of both wastes resulted in higher production of endoinulinase than either waste alone. Also, ten fungal species grown on these substrates as inexpensive, carbon sources were screened for the best production of endoinulinase activities. Of these, Aspergillus niger AUMC 9375 was the most productive, when grown on the mixture using a 6:1 w/w ratio of sun flower: lettuce, and yielded the highest levels of inulinase at 50% moisture, 30°C, pH 5.0, with seven days of incubation, and with yeast extract as the best nitrogen source. Inulinase was purified to homogeneity by ion-exchange chromatography and gel-filtration giving a 51.11 fold purification. The mixture of sunflower tubers and lettuce roots has potential to be an effective and economical substrate for inulinase production. Inulinase was successfully immobilized with an immobilization yield of 71.28%. After incubation for 2 h at 60°C, the free enzyme activity decreased markedly to 10%, whereas that of the immobilized form decreased only to 87%. A reusability test demonstrated the durability of the immobilized inulinase for 10 cycles and in addition, that it could be stored for 32 days at 4°C. These results indicate that this inulinase, in the immobilized form, is a potential candidate for large-scale production of high purity fructose syrups.