Immobilization of Enzyme on nanoparticles

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.

Lactoperoxidase (LPO) is a glycoprotein enzyme with a wide antimicrobial activity. Its stabilization is a key
instrument for use LPO in industry. Gum tragacanth is a biopolymer, which is used for encapsulation and
chitosan is used as a matrix for protein immobilization. This paper attempts to immobilize LPO by tragacanthchitosan
nano-biopolymers. Start with enzyme activity evaluating, LPO loading was done after analyzing
tragacanth-chitosan nanocomposites by zeta sizer, FT-IR and SEM. Determining the nanocomposites proper
size, the stability of immobilized and free LPO during storage was evaluated after 14 days of storage in 4 °C and
25 °C. Thermal stability also estimated by storing the immobilized and free LPO in different temperatures from 0
to 80 °C. It was found that, using nanocomposites with the size of 356.8 nm would result to preserving more
stability during storage. The immobilized enzyme would have also more thermal stability. The results may be the
consequences of encapsulation of LPO in tragacanth-chitosan nano-biopolymers. These findings suggest the
application of tragacanth-chitosan nano-biopolymers in LPO immobilizing.

Enzyme immobilization often achieves reusable biocatalysts with improved operational stability and solvent resistance. However, these modifications are generally associated with a decrease in activity or detrimental modifications in catalytic properties. On the other hand, protein engineering aims to generate enzymes with increased performance at specific conditions by means of genetic manipulation, directed evolution and rational design. However, the achieved biocatalysts are generally generated as soluble enzymes, −thus not reusable-and their performance under real operational conditions is uncertain. Combined protein engineering and enzyme immobilization approaches have been employed as parallel or consecutive strategies for improving an enzyme of interest. Recent reports show efforts on simultaneously improving both enzymatic and immobilization components through genetic modification of enzymes and optimizing binding chemistry for site-specific and oriented immobilization. Nonetheless, enzyme engineering and immobilization are usually performed as separate workflows to achieve improved biocatalysts. In this review, we summarize and discuss recent research aiming to integrate enzyme immobilization and protein engineering and propose strategies to further converge protein engineering and enzyme immobilization efforts into a novel "immobilized biocatalyst engineering" research field. We believe that through the integration of both enzyme engineering and enzyme immobilization strategies, novel biocatalysts can be obtained, not only as the sum of independently improved intrinsic and operational properties of enzymes, but ultimately tailored specifically for increased performance as immobilized biocatalysts, potentially paving the way for a qualitative jump in the development of efficient, stable biocatalysts with greater real-world potential in challenging bioprocess applications.

(FTases) (EC 2.4.1.9). However, FTase reuse is meagre and the cost of the enzyme production is high, making FOS production economically unfeasible. In order to address these issues, this study aims to immobilize cells from Aspergillus oryzae IPT-301, containing FTase enzyme previously glutaraldehyde-crosslinked, by means of entrapment in calcium alginate beads. The conditions to obtain the particles, such as alginate type, mycelium mechanical dispersion, degree of crosslinking, size of the beads, and their reflections on transfructosylation activity (AT) were evaluated previously.
RESULTS: The highest AT used low-viscosity alginate solution (4.0% w/w) crosslinked with calcium chloride (0.2 mol L−1) and beads of mean diameter 2.15 mm. The entrapment conditions were complemented by conducting the reaction under different temperatures, pH values, substrate concentrations and by reusing the beads. The highest transfructosylation activities were obtained under reaction conditions of 50 °C, pH 5.5 and substrate concentration 400 g L−1.
CONCLUSION: The AT levels found with the glutaraldehyde-crosslinked and entrapped cells in calcium alginate were ≈70% higher than the ones obtained with free cells, not glutaraldehyde-crosslinked. Moreover, the immobilized cells (crosslinked +entrapped) retained their AT up to the third cycle of total sucrose depletion in the reaction medium, attesting to the potential use of the beads in continuous FOS production.

The process described in the present work uses air supplementation in a flui-dized bed reactor containing Bacillus firmus strain 37 immobilized on active bovine bone charcoal, to produce by batch fermentation the enzyme CGTase (cyclomaltodextrin-glucanotransferase). Three different aeration rates were evaluated. The maximum CGTase activity was achieved after 120 hours of fermentation with aeration rate of 2 vvm and was equal to 2.48 U/mL. When 0.5 and 1 vvm were used the enzymatic activities achieved 1.1 and 0.57 U/mL, respectively. Bovine bone charcoal was characterized in terms of surface area, pore size and volume. To the best of our knowledge, the immobilization of microorganism cells in bovine bone charcoal for CGTase production has not been reported in the literature. Our results showed that fluidized bed reactor allows retaining high concentration of biomass, improving biomass-substrate contact and operation at low residence times, which resulted in improved enzyme production. Therefore, the process as proposed has great potential for industrial development.

Aims:  To study protease production from four Bacillus species.
To optimize the production of extracellular protease by testing
various environmental and nutritional factors and its application as
a contact lens cleanser.
Methodology: The assay was carried out in duplicates. Cultures of
Bacillus subtilis, Batcillus licheniformis, Bacillus thermophilus,
and  Bacillus cereus  were used for Protease production and assay.
Sterile Bushnell  –  Hass medium containing 1% casein was used.
The clear zone of casein hydrolysis indicated protease secretion.
Protease assay was done using modified sigma protocol for
enzymatic assay of protease. Effect of incubation time, pH,
temperature and aeration on protease production was studied.
Diameter of colony and zone of clearance was measured and Cx
ratio was calculated. The best protease producer was selected.
Extracted and crude protease enzymes were added to artificial tear
solution prepared with 0.2% lysozyme. Enzyme treatment was
done for 10, 30, 60 and 90 min at optimum temperatures of
respective enzymes. Light  transmission readings were recorded
using visible range spectrophotometer at 285 nm.
Results:  All four species of Bacillus produced protease enzyme.
Bacillus subtilis (Cx ratio=6) was the best protease producer. 37
o
C
temperature, 48 hrs of incubation and pH 7.5 was optimum for
maximum production of protease from Bacillus subtilis. Optimum
temperature and pH for the crude enzyme activity was 40ºC and 8.
The optimum ammonium sulfate fractionation (40% (w/v)
saturation) showed 4.76 fold increase in the specific activity of the
crude extract. Protein solution was degraded by purified enzyme in
30 min whereas crude enzyme required 60 min.
Conclusion:  Protease enzyme extracted from  Bacillus subtilis
showed good activity against artificial tear solution.

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 intermolecular force measurement of the binding of the two partners of the split intein (called protein transsplicing). 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 1 μm/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 nonpolar 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.

In this work, we developed a new amperometric biosensor for glutamate detection using a typical method of glutamate oxidase (GlOx) immobilization via adsorption on silicalite particles. The disc platinum electrode (d = 0.4 mm) was used as the amperometric sensor. The procedure of biosensor preparation was optimized. The main parameters of modifying amperometric transducers with a silicalite layer were determined along with the procedure of GlOx adsorption on this layer. The biosensors based on GlOx adsorbed on silicalite demonstrated high sensitivity to glutamate. The linear range of detection was from 2.5 to 450 μM, and the limit of glutamate detection was 1 μM. It was shown that the proposed biosensors were characterized by good response reproducibility during hours of continuous work and operational stability for several days. The developed biosensors could be applied for determination of glutamate in real samples.

Lactoperoxidase (LPO) is a glycoprotein enzyme with a wide antimicrobial activity. Its stabilization is a key instrument for use LPO in industry. Gum tragacanth is a biopolymer, which is used for encapsulation and chitosan is used as a matrix for protein immobilization. This paper attempts to immobilize LPO by tragacanthchitosan nano-biopolymers. Start with enzyme activity evaluating, LPO loading was done after analyzing tragacanth-chitosan nanocomposites by zeta sizer, FT-IR and SEM. Determining the nanocomposites proper size, the stability of immobilized and free LPO during storage was evaluated after 14 days of storage in 4 °C and 25 °C. Thermal stability also estimated by storing the immobilized and free LPO in different temperatures from 0 to 80 °C. It was found that, using nanocomposites with the size of 356.8 nm would result to preserving more stability during storage. The immobilized enzyme would have also more thermal stability. The results may be the consequences of encaps...

The process described in the present work uses air supplementation in a flui-dized bed reactor containing Bacillus firmus strain 37 immobilized on active bovine bone charcoal, to produce by batch fermentation the enzyme CGTase (cyclomaltodextrin-glucanotransferase). Three different aeration rates were evaluated. The maximum CGTase activity was achieved after 120 hours of fermentation with aeration rate of 2 vvm and was equal to 2.48 U/mL. When 0.5 and 1 vvm were used the enzymatic activities achieved 1.1 and 0.57 U/mL, respectively. Bovine bone charcoal was characterized in terms of surface area, pore size and volume. To the best of our knowledge, the immobilization of microorganism cells in bovine bone charcoal for CGTase production has not been reported in the literature. Our results showed that fluidized bed reactor allows retaining high concentration of biomass, improving biomass-substrate contact and operation at low residence times, which resulted in improved enzyme production...

The process described in the present work uses air supplementation in a fluidized bed reactor containing Bacillus firmus strain 37 immobilized on active bovine bone charcoal, to produce by batch fermentation the enzyme CGTase (cyclomaltodextrin-glucanotransferase). Three different aeration rates were evaluated. The maximum CGTase activity was achieved after 120 hours of fermentation with aeration rate of 2 vvm and was equal to 2.48 U/mL. When 0.5 and 1 vvm were used the enzymatic activities achieved 1.1 and 0.57 U/mL, respectively. Bovine bone charcoal was characterized in terms of surface area, pore size and volume. To the best of our knowledge, the immobilization of microorganism cells in bovine bone charcoal for CGTase production has not been reported in the literature. Our results showed that fluidized bed reactor allows retaining high concentration of biomass, improving biomass-substrate contact and operation at low residence times, which resulted in improved enzyme production. Therefore, the process as proposed has great potential for industrial development.

In this work, the method of enzyme adsorption on different zeolites and mesoporous silica spheres (MSS) was investigated for the creation of conductometric biosensors. The conductometric transducers consisted of gold interdigitated electrodes were placed on the ceramic support. The transducers were modified with zeolites and MSS, and then the enzymes were adsorbed on the transducer surface. Different methods of zeolite attachment to the transducer surface were used; drop coating with heating to 200°C turned out to be the best one. Nanozeolites beta and L, zeolite L, MSS, and silicalite-1 (80 to 450 nm) were tested as the adsorbents for enzyme urease. The biosensors with all tested particles except zeolite L had good analytical characteristics. Silicalite-1 (450 nm) was also used for adsorption of glucose oxidase, acetylcholinesterase, and butyrylcholinesterase. The glucose and acetylcholine biosensors were successfully created, whereas butyrylcholinesterase was not adsorbed on silicalite-1. The enzyme adsorption on zeolites and MSS is simple, quick, well reproducible, does not require use of toxic compounds, and therefore can be recommended for the development of biosensors when these advantages are especially important.

Rat liver mitochondria are shown to adhere to the alkylsilylated glass beads in essentially a monolayer. The amount of mitochondria bound to the beads reaches a maximum where the length of the alkyl groups covalently linked to the beads exceeds eight carbons. Mitochondria immobilized on the beads and placed in a flow system exhibit normal: (a) respiratory control, (b) phosphate to oxygen ratio, (c) uncoupling by 2,4-dinitrophenol and carbonylcyanide p-trifluoromethoxyphenylhydrazone, and (d) inhibition by cyanide, azide, rotenone, oligomycin, and antimycin. Reversibility of the effects of 2,4-dinitrophenol, cyanide, and azide was rapid and complete. Inhibition by rotenone, oligomycin was essentially irreversible. Mitochondria have been maintained in a viable state on the beads at 27° for periods up to 4 hours. The use of immobilized organelles appears to offer a new technique for the study of membrane-bound particles whereby substances can be rapidly added and removed while monitoring the composition of solution flowing over the particles.