Carrier For Enzyme Immobilization Research Articles

Immobilizing enzymes in regular-sized gelatin microspheres through a membrane emulsification method

Enzyme immobilization is a promising biotechnology which not only ensures reusability of enzymes and permits easy biocatalyst-product separation, but also improves the storage stability of enzymes. It overcomes many drawbacks of free enzyme, and hence presents significant applications in bioscience and bioengineering. Microspheres composed of biopolymer hydrogel are widely used as carriers for enzyme immobilization. In this study, the regular-sized gelatin microspheres were prepared using the membrane emulsification method, and were subsequently utilized for the encapsulation of yeast alcohol dehydrogenase (YADH). The results indicated that the recycling stability, pH tolerance, and storage stability of the immobilized enzyme are all significantly improved. The immobilized YADH maintained higher relative activity than its free form under extreme acidic conditions down to pH 4.0 (63 vs. 16 %) and alkaline conditions up to pH 10.0 (42 vs. 0 %). Additionally, microspheres of different sizes can be prepared using membranes of different pore sizes. The further experiment established a linear correlation between the average diameter of microspheres and the pore size of membranes. The effects of preparation conditions on the diameter and uniformity of microspheres were also investigated systematically.

Conferring Natural-Derived Porous Microspheres with Surface Multifunctionality through Facile Coordination-Enabled Self-Assembly Process.

In this study, multifunctional chitin microspheres are synthesized and utilized as a platform for multiple potential applications in enzyme immobilization, catalytic reduction and adsorption. Porous chitin microspheres with an average diameter of 111.5 μm and a porous architecture are fabricated through a thermally induced phase separation method. Then, the porous chitin microspheres are conferred with surface multifunctionality through facile coordination-enabled self-assembly of tannic acid (TA) and titanium (Ti(IV)) bis(ammonium lactate)dihydroxide (Ti-BALDH). The multipoint hydrogen bonds between TA and chitin microspheres confer the TA-Ti(IV) coating with high adhesion capability to adhere firmly to the surface of the chitin microspheres. In view of the biocompatibility, porosity and surface activity, the multifunctional chitin microspheres are used as carriers for enzyme immobilization. The enzyme-conjugated multifunctional porous microspheres exhibit high catalytic performance (102.8 U·mg(-1) yeast alcohol dehydrogenase). Besides, the multifunctional chitin microspheres also find potential applications in the catalytic reduction (e.g., reduction of silver ions to silver nanoparticles) and efficient adsorption of heavy metal ions (e.g., Pb(2+)) taking advantages of their porosity, reducing capability and chelation property.

Synthesis of butyl butyrate in 93 % yield by Thermomyces lanuginosus lipase on waste eggshells

There is a high demand for immobilized enzymes in the industry because it is a green technology, it improves the efficiency of the crude enzyme and it minimizes the step of product recovery. Selection of a suitable carrier for enzyme immobilization is crucial. Eggshells are abundantly available as an organic waste. Here, lipase from Thermomyces lanuginosus was immobilized on eggshell to produce butyl butyrate. We evaluated the effect of lipase loading, from 0.5 to 3.0 %, size of eggshells, lower than 1, 1–2 and 2–4 mm, and temperature, from 30 to 50 °C. Results show that the optimum conditions calculated by response surface methodology analysis for butyl butyrate synthesis were 40 % w/v of immobilized lipase loading and 0.2 M of substrate concentration at 38 °C during 24 h. The predicted yield was 92.78 %, and the yield obtained during synthesis was 93.48 %.

Layer-by-Layer Assembly of Metal-Organic Frameworks in Macroporous Polymer Monolith and Their Use for Enzyme Immobilization.

New monolithic materials comprising zeolitic imidazolate framework (ZIF-8) located on the pore surface of poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith previously functionalized with N-(3-aminopropyl)-imidazole have been prepared via a layer-by-layer self-assembly strategy. These new ZIF-8@monolith hybrids are used as solid-phase carriers for enzyme immobilization. Their performance is demonstrated with immobilization of a model proteolytic enzyme trypsin. The best of the conjugates enable very efficient digestion of proteins that can be achieved in mere 43 s.

Modified nanoporous titanium dioxide as a novel carrier for enzyme immobilization

ε-Poly-l-lysine (EPL)-modified mesoporous titanium dioxide (M-TiO2) was assembled through the electrostatic attraction between EPL and M-TiO2. Through modification, the M-TiO2 surface tends to form multilayered and complex architectures, which can be used as artificial matrices to change the microenvironment of carriers for enzyme immobilization. The modified M-TiO2 was characterized through scanning electron microscopy, Fourier transform infrared spectroscopy, zeta potential analysis, and thermogravimetric analysis. All of the immobilized enzymes with negative charges display strong storage stability, thermal stability, and good reusability. Results indicate that EPL can self-assemble onto the surface of M-TiO2 and form a considerable number of active coatings. Our results also demonstrate that this simple and novel method can be potentially used to immobilize negatively charged enzymes for biosensor applications.

Immobilized rennin in TC/SG composite in cheese production

The object of the current study was to develop a new process for continuous Feta-type cheese production using a biocatalyst consisting of immobilized rennin on a tubular cellulose/starch gel (TC/SG) composite, which has been proven to be an appropriate carrier for enzyme immobilization. Different methodologies were used in order to prepare four biocatalysts. The most effective was selected for cheese production in a 1L continuous system, providing two economically useful results for the dairy industries: (i) increase of productivity by the continuous coagulation of milk, and (ii) saving of the rennin enzyme expenses of the batch coagulation of milk. The criteria used to choose the appropriate biocatalyst was based on the time of coagulation in successive batches, the concentration of immobilized rennin combined with the filter efficiency and its application in the continuous system. Physicochemical analyses of the cheeses at various stages of the ripening were performed. No significant differences compared to cheeses prepared with the traditional method were found. Aroma compounds were determined by SPME GC–MS.

Magnetic AuNP@Fe3O4 nanoparticles as reusable carriers for reversible enzyme immobilization

A reusable carrier for enzyme immobilization consisting of magnetic Fe3O4 nanoparticles coated with a layer of gold nanoparticles (AuNPs) has been demonstrated. The gold nanoparticles served as an intermediate ligand on which trypsin was reversibly immobilized. This immobilized trypsin reactor was used for the digestion of standard proteins that was achieved within 15min. After digestion, the trypsin-loaded nanoparticles were recovered from the reaction mixture using a magnet. The peptides in digest were then determined using HPLC and MALDI-TOF mass spectrometry. Sequence coverages of 93.8%, 90.9%, and 54.1% were observed for myoglobin, cytochrome c, and bovine serum albumin, respectively. Our newly designed AuNP@Fe3O4 enzymatic nanosystem enabled significant acceleration of the enzyme catalyzed reaction. We also observed a higher efficiency compared with in-solution digestion using free trypsin, and with reactors produced using conventional immobilization techniques such as electrostatic interaction, covalent bonding, and formation of metal chelate complexes. Furthermore, the use of gold nanoparticles as the intermediate ligand enabled regeneration of the enzyme and recovery of the activity in our system simply by stripping the inactive trypsin from the gold nanoparticles using sodium borohydride followed by the immobilization of fresh trypsin.

Editorial

Biotechnology has become an important area for the production of smart materials under environmentally friendly conditions. On the one hand, microorganisms can produce polymers where novel strategies in biotechnology allow tuning of their composition and consequently their properties. On the other hand, enzymes are increasingly used both to replace toxic chemicals in processing of materials as well as for targeted surface modification. The detailed characterization of microbial polymers and the screening for their producers is an important aspect in this context which is therefore carefully reviewed in this issue for the most promising candidates (e.g. polyhydroxyalcanoates, PHAs). In another contribution, cheap substrates for the production of microbial polymers such as fermentation effluents have been evaluated. In terms of the perspective applications of these materials, the potential of PHAs for Peripheral Nerve Regeneration has been assessed in this issue. For what concerns polysaccharides, other authors have investigated the photoprotective and antioxidant properties of chitosan containing nanoemulsions. Apart from giving access to important medical and cosmetic applications, biotechnology is gaining increasing importance in bulk fiber processing. Here examples are given related to the exploitation of novel fibres from bamboo obtained via enzymatic retting. In turn, textiles can serve as excellent carriers for enzyme immobilization. The editors would like to thank all authors for their excellent contributions and the reviewers for their help in the preparation of this issue. Sincerely, Artur Cavaco-Paulo Universidade de Minho, Centre of Biological Engineering Gianluca Ciardelli Politecnico di Torino, Department of Mechanics Georg M. Gübitz University of Natural Resources and Life Sciences, Austria Institute for Environmental Biotechnology

Influence of compression speed and deformation percentage on mechanical properties of calcium alginate particles

Hydrogel particles are often used as a carrier for immobilization of enzymes, polyphenolic antioxidants, whole microbial, plant or mammalian cells. In many processes, mechanical properties of alginate particles are essential due to their exposure to mechanical forces in production process. Determination and improvement of hydrogels mechanical properties is very important in prevention of the undesirable side effects during the manufacturing process and product application. The aim of this study was to define the mechanical properties of single particles submerged in water and in dry conditions using the compression method between two flat surfaces. The results indicated that the formulation of alginate beads and water loss during compression have significant influence on their mechanical behavior and stiffness. Calcium-alginate particles were produced using an electrostatic droplet extrusion technique, with an initial sodium alginate concentration of 1.5%w/v and calcium chloride (2.0% w/v) as gelling solution. The research findings were used to determine the influence of working conditions, sample deformation (10-50%) and different compression speeds (1-50mm/min) on mechanical strength of alginate beads. The Young?s modules and maximal forces for investigated deformation percentage of the alginate particles were determined from generated force-displacement and stress-strain curves during compression.

Rational synthesis of novel recyclable Fe₃O₄@MOF nanocomposites for enzymatic digestion.

In this work, for the first time, magnetic MOFs were synthesized as carriers for enzyme immobilization. The magnetic MOF-enzyme composites exhibited significantly improved digestion performance, excellent reusability, stability and reproducibility for different kinds of enzymes.

Fabrication and characterization of core-shell magnetic chitosan nanoparticles as a novel carrier for immobilization of Burkholderia cepacia lipase.

In this study, the chitosan magnetic core-shell nanoparticles (CMNPs) was synthesized and then used as a support for immobilization of lipase. The characteristics of CMNPs, including morphology, topography and spectra type before and after immobilization were determined. The scanning electron micrographs of the CMNPs showed that they were approximately uniform spheres and the distribution chart indicated that the particles have the mean diameter of 100 nm. Kinetic parameters of Km and Vm were calculated as 1.07 mM and 29.43 U/mg for free B. cepacia lipase and 1.29 mM and 25.82 U/mg for immobilized lipase on CMNPs, respectively. The activity of immobilized lipase was 32 U/mg under optimum temperature and pH. CMNP's were used in trasesterification reaction in order to evaluate the activity of the immobilized enzyme compared to the free enzyme. Immobilization of lipase on CMNPs improved stability and total relative activity of the enzyme. It could be concluded that CMNPs be considered as a suitable carrier for enzyme immobilization.

Exceptional thermal stability of industrially-important enzymes by entrapment within nano-boehmite derived alumina

We developed an alumina sol–gel matrix based on boehmite nanorods as a superior carrier for enzyme immobilization.

A mild strategy to encapsulate enzyme into hydrogel layer grafted on polymeric substrate.

Although the hydrogel network has been widely investigated as a carrier for enzyme immobilization, to in situ encapsulate enzymes into a hydrogel network in an efficient, practical, and active way is still one of the great challenges in the field of biochemical engineering. Here, we report a new protocol to address this issue by encapsulating enzyme into poly(ethylene glycol) (PEG) hydrogel network grafted on polymeric substrates. In our strategy, isopropyl thioxanthone semipinacol (ITXSP) dormant groups were first planted onto the surface of a plastic matrix with low density polyethylene (LDPE) film as a model by a UV-induced abstracting hydrogen-coupling reaction. As a proof of concept, lipase, which could catalyze esterification of glucose with palmitic acid, then was in situ net-immobilized into a PEG-based hydrogel network layer through a visible light-induced surface controlled/living graft cross-linking polymerization. This strategy demonstrates the following novel significant merits: (1) in comparison with the UV irradiation or high temperature, the visible light and room temperature used provide a friendly condition to maintain activity of enzyme during immobilization; (2) the uniqueness of controlled/living cross-linking polymerization not only makes it easy to form a uniform PEG hydrogel network, which is a benefit to avoid the leakage of net-immobilizing enzyme, but also to tune the net-thickness or capacity to accommodate enzyme; and (3) as compared to systems of nanoparticles and porous matrixes, the flexible/robust end-products of the surface net-immobilizing enzyme with polymer film are more suitable to be applied in a bioreactor due to their features of easier separation and reuse. We confirmed that this catalytic film could retain almost all of its initial activity after seven batches of 24 h esterifications. The proposed strategy provides an extremely simple, effective, and flexible method for enzyme immobilization.

Immobilization of Aspergillus terreus lipase in self-assembled hollow nanospheres for enantioselective hydrolysis of ketoprofen vinyl ester

The aim of this study was to improve the ability of Aspergillus terreus lipase to separate the racemic ketoprofen vinyl ester into individual enantiomers using hollow self-assembly alginate-graft-poly(ethylene glycol)/α-cyclodextrins (Alg-g-PEG/α-CD) spheres as enzyme immobilization carriers. The morphology and size of the Alg-g-PEG/α-CD particles were investigated by transmission electron microscopy (TEM) and were found to be nanoscale. To facilitate recycling, calcium alginate (CA) beads were developed to encapsulate Alg-g-PEG/α-CD particles, thereby producing Alg-g-PEG/α-CD/CA composite beads. The influence of buffer pH and enzyme concentration during immobilization was studied along with the biocatalyst's kinetic parameters. When the immobilized enzyme was under optimal conditions in the resolution reaction, maximal conversion (approximately 45.9%) and enantioselectivity (approximately 128.8) were obtained. The immobilized A. terreus lipase maintained excellent performance even after 20 reuses and retained nearly 100% of its original activity after 24 weeks of storage at 4°C.

Poly(2-vinyl-4,4-dimethylazlactone)-functionalized magnetic nanoparticles as carriers for enzyme immobilization and its application.

Fabrication of various efficient enzyme reactors has triggered increasing interests for its extensive applications in biological and clinical research. In this study, magnetic nanoparticles were functionalized by a biocompatible reactive polymer, poly(2-vinyl-4,4-dimethylazlactone), which was synthesized by reversible addition-fragmentation chain transfer polymerization. Then, the prepared polymer-modified magnetic nanoparticles were employed as favorable carriers for enzyme immobilization. l-Asparaginase was selected as the model enzyme to fabricate the enzyme reactor, and the prepared enzyme reactor exhibited high loading capacity of 318.0 μg mg(-1) magnetic nanoparticle. Interestingly, it has been observed that the enzymolysis efficiency increased slightly with the lengthened polymer chain, resulting from the increased immobilization amount of enzyme. Meanwhile, the immobilized enzyme could retain more than 95.7% activity after 10 repeated uses and maintain more than 72.6% activity after 10 weeks storage. Moreover, an extracorporeal shunt system was simulated to estimate the potential application capability of the prepared l-asparaginase reactor in acute lymphoblastic leukemia treatment.

POTENTIALITIES OF ZEOLITES FOR IMMOBILIZATION OF ENZYMES IN CONDUCTOMETRIC BIOSENSORS

Conductometric biosensors based on urease and glucose oxidase immobilized with different types of zeolites have been investigated and compared. For this purpose, zeolite A, zeolite Y, Silicalite-1 (spherical), Silicalite-2, H+Beta 300, H+Beta 150 and NH4+Beta 25 were compared as potential carriers for enzyme immobilization. The parameters to obtain optimized biosensor performance were studied by investigating the percentage of zeolite in membrane, immobilization time in glutaraldehyde vapor and pH of the environment. Different zeolite types resulted in different enzymatic responses. In particular, we have demonstrated that the urease immobilized on silicalite-2 had better performance than immobilized urease without zeolite. Conductometric biosensor with glucose oxidase immobilized with NH4+Beta 25 zeolite had similar response values compared with immobilised enzyme without zeolite. The results obtained show that zeolites could be used as alternatives for enzyme immobilization in conductometric biosensors development.

Applied usage of yeast spores as chitosan beads.

In this study, we present a nonhazardous biological method of producing chitosan beads using the budding yeast Saccharomyces cerevisiae. Yeast cells cultured under conditions of nutritional starvation cease vegetative growth and instead form spores. The spore wall has a multilaminar structure with the chitosan layer as the second outermost layer. Thus, removal of the outermost dityrosine layer by disruption of the DIT1 gene, which is required for dityrosine synthesis, leads to exposure of the chitosan layer at the spore surface. In this way, spores can be made to resemble chitosan beads. Chitosan has adsorptive features and can be used to remove heavy metals and negatively charged molecules from solution. Consistent with this practical application, we find that spores are capable of adsorbing heavy metals such as Cu(2+), Cr(3+), and Cd(2+), and removal of the dityrosine layer further improves the adsorption. Removal of the chitosan layer decreases the adsorption, indicating that chitosan works as an adsorbent in the spores. Besides heavy metals, spores can also adsorb a negatively charged cholesterol derivative, taurocholic acid. Furthermore, chitosan is amenable to chemical modifications, and, consistent with this property, dit1Δ spores can serve as a carrier for immobilization of enzymes. Given that yeast spores are a natural product, our results demonstrate that they, and especially dit1Δ mutants, can be used as chitosan beads and used for multiple purposes.

Facile preparation of magnetically functionalized graphite nanosheets for porcine pancreatic lipase immobilization

The graphite nanosheets decorated with Fe3O4 nanoparticles (about 15–20 nm), i.e., Fe3O4@graphite nanosheets, have been prepared via a facile one-pot method. The resulting composites with large specific surface area, high affinity, and rapid magnetic response were characterized by means of Fourier transform infrared spectra, Raman spectra, powder X-ray diffraction, scanning electron microscopy, transmission electron microscopy, elemental analysis, and thermogravimetric analysis, which indicated that the Fe3O4 nanoparticles were chemically integrated on the graphite nanosheets. The porcine pancreatic lipase (PPL) was immobilized onto the Fe3O4@graphite nanosheets through covalent bonding using glutaraldehyde as a coupling reagent, and the properties of the resulting immobilized PPL such as enzymatic activity, stability, and reusability were researched at the same time. The result proved that the immobilized PPL displayed excellent properties and demonstrated that the prepared Fe3O4@graphite nanosheets would present a potential application as a novel carrier for enzyme immobilization, enrichment, and separation.

Study on Poly(ether imide)/Amino-Modified Sillica Hybrid Membranes

Poly (ether imide) (PEI) membrane with many good properties, such as chemical resistance, biocompatibility, good thermal, and mechanical properties, has been widely used in filtration and biomedical applications. SiO2particles containing amine groups can be a good enzyme immobilization carrier. Organic-inorganic hybrids of poly (ether imide) and amino-modified SiO2were in situ prepared via sol-gel process in the presence of tetraethylortho silicate (TEOS) in a mixed solvent of N, N-Dimethylacetamide/1-Methyl-2-pyrrolidone (DMAc/NMP). The effects of casting solution composition on the membrane morphological structure and performance were investigated. The dispersion of the inorganic and organic phase in the nanoscale was possible with addition of (3-aminopropyl) triethoxysilane (APTES) as shown by field emission electron microscopy. Membranes with different ratio of APTES/TEOS have quite different properties. Mechanical properties evaluation such as rupture elongation ration and tensile strength tests of the hybrid membranes were carried out. Fourier transform infrared spectroscopy (FTIR), pure water fluxes and water contact angle of the hybrid membranes were also characterized. The results show that poly (ether imide)/amino-modified SiO2hybrid membranes have high static adsorption capacity for lysozyme (Lys). Moreover, mechanical properties of the hybrid membranes greatly improve compared to pure PEI membranes.

Nylon 6 film and nanofiber carriers: Preparation and laccase immobilization performance

Enzyme immobilization has attracted continuous attention in the field of fine chemistry, biomedicine and biosensor. Polyamides are promising materials to promote immobilization: thus, nylon 6 film and nanofiber carriers (prepared by electrospinning method) have been investigated. The enzyme (i.e. laccase from Trametes versicolor) was covalently immobilized onto spacer-arm attached carriers after acidic hydrolysis. The amount of immobilized enzyme on the nylon film and nanofibers was 59.4% and 71.0% respectively. The maximum activity (Vmax) and Michaelis–Menten constant (Km) of laccase immobilized on functionalized carriers were determined. The operational and thermal stabilities of the immobilized laccase were improved compared to free counterpart. The use of nylon carriers for enzyme immobilization has shown interesting properties to be used as biocatalytic material in industrial applications. Furthermore, nylon carriers are cheap and could be produced at large scale.