Immobilization Of L-asparaginase Research Articles

Immobilization of L-Asparaginase on biofunctionalized magnetic graphene oxide nanocomposite: A promising approach for Enhanced Stability and reusability

Immobilization of L-Asparaginase on biofunctionalized magnetic graphene oxide nanocomposite: A promising approach for Enhanced Stability and reusability

Immobilization of l-asparaginase on genipin cross-linked chitosan beads shows better acrylamide diminution in cassava chips: Process optimization and characterization.

Glutaraldehyde is the conventionally used cross-linker for the activation and cross-linking of support matrices used in enzyme immobilization. However, the toxic nature of glutaraldehyde makes it unsafe for food applications, propelling the need for nontoxic cross-linkers. Genipin reacts with the primary and secondary amines generating a dark-blue colored pigment and is an attractive alternative to glutaraldehyde as a cross-linker for enzyme immobilization. Apart from its excellent cross-linking properties, genipin possesses added advantages over glutaraldehyde such as proven health benefits, biocompatibility, and biodegradability. The present study explores the application of chitosan beads cross-linked with the natural and nontoxic agent, genipin, for immobilizing l-asparaginase, aimed at its subsequent use in mitigating acrylamide formation in food products. The immobilized l-asparaginase exhibited improved functionalities such as stability, reusability, and reduction in acrylamide formation in deep-fried cassava chips. One of the limitations observed during application in the food process was the mechanical fragility of the chitosan beads during speedy stirring. This can be overcome by increasing the concentration and time of contact of the coagulant bath during the formation of chitosan beads. The drying of the enzyme-bound chitosan beads will also lead to shrinkage and prevent breakage during stirring. This study conclusively demonstrated the applicability of immobilizing l-asparaginase on genipin cross-linked chitosan beads in food-related processes.

Immobilization of L-asparaginase on Oxidized Bacterial Cellulose to Improve the Thermal Stability of the Enzyme

Bacterial cellulose (BC) membranes can be modified for covalent immobilization of macromolecules. One type of modification is oxidation, after which the oxidized BC membrane (OBC) could be used as a matrix for covalent immobilization of enzymes. In this work, the BC membrane was chemically oxidized with sodium periodate (NaIO4) to increase the stability of immobilized mesophilic L-asparaginase (L-ASNase) from Erwinia carotovora (EwA). IR spectroscopy confirmed the immobilization of L-ASNase EwA on OBC membranes. Immobilization of the enzyme increased its temperature optimum for its activity by 15°C and raised the inactivation temperature to 60°C. The OBC membrane could be used as a potential carrier for covalent immobilization of enzymes to improve their pharmacological properties by increasing their thermostability.

Immobilization of l-asparaginase on chitosan nanoparticles for the purpose of long-term application

Asparaginase holds significant commercial value as an enzyme in the food and pharmaceutical industries. This study examined the optimum and practical use of the l-asparaginase derived from Pseudomonas aeruginosa HR03. Specifically, the study focused on the effectiveness of the stabilized enzyme when applied to chitosan nanoparticles. The structure, size, and morphology of chitosan nanoparticles were evaluated in relation to the immobilization procedure. This assessment involved the use of several analytical techniques, including FT-IR, DLS, SEM, TEM, and EDS analysis. Subsequently, the durability of the enzyme that has been stabilized was assessed by evaluating its effectiveness under extreme temperatures of 60 and 70 °C, as well as at pH values of 3 and 12. The findings indicate that incorporating chitosan nanoparticles led to enhanced immobilization of the l-asparaginase enzyme. This improvement was observed in terms of long-term stability, stability under crucial temperature and pH conditions, as well as thermal stability. In addition, the optimum temperature increased from 40 to 50 °C, and the optimum pH increased from 8 to 9. Enzyme immobilization led to an increase in Km and a decrease in kcat compared to its free counterpart. Because of its enhanced long-term stability, l-asparaginase immobilization on chitosan nanoparticles may be a potential choice for use in industries that rely on l-asparaginase enzymes, particularly the pharmaceutical and food industries.

Enhancing biotechnological applications of l-asparaginase: Immobilization on amino-epoxy-agarose for improved catalytic efficiency and stability

l-asparaginase has been shown to be effective in reducing acrylamide formation in food and in treating certain cancers, such as acute lymphoblastic leukemia. This study aimed to improve the application of l-asparaginase in these areas, through its immobilization in two distinct supports: epoxy-agarose and amino-epoxy-agarose. Different immobilization conditions were investigated, including immobilization pH and buffer concentration. The best activity results were obtained employing amino-epoxy-agarose, being the 0.1 M sodium carbonate buffer pH 10 the conditions that offered the highest immobilization yield (76%), immobilization efficiency (89.8%) and relative activity (68.2%). Immobilization of l-asparaginase in amino-epoxy-agarose improved its catalytic efficiency and stability, including at pH 4 and 60 °C. All immobilized biocatalysts preserved more than 70% of their activity after 7 cycles of reuse, and maintained about 82% of their activity after 60 days of storage at 4°C. These results show the efficacy of immobilized biocatalysts in the preservation of enzymatic activity and highlight their potential for biotechnological applications.

Immobilization of L-asparaginase on magnetic nanoparticles: Kinetics and functional characterization and applications

L-asparaginase shows great potential as a food enzyme to reduce acrylamide formation in fried and baked products. But for food applications, enzymes must be stable at high temperatures and have higher catalytic efficiency. These desirable characteristics are conferred by the immobilization of enzymes on a suitable matrix. The present study aimed to immobilize the L-asparaginase enzyme on magnetic nanoparticles to reduce acrylamide content in the food system. Immobilized preparations were characterized using SEM, TEM, FTIR, UV-spectrometry, and XRD diffraction analyses. These nanoparticles enhanced the thermal stability of the enzyme up to four-fold at 70 °C compared to the free enzyme. Kinetic parameters exhibited an increase in Vmax, Km, and catalytic efficiency by ~ 38% than the free counterpart. The immobilized preparations were reusable for up to five cycles. Moreover, their application in the pre-treatment coupled with blanching of potato chips led to a significant reduction (greater than 95%) of acrylamide formation.

Study on controllable enzymolysis by chiral capillary electrophoresis with an ultraviolet-visible responsive polymer membrane based l-asparaginase reactor

Stimuli-responsive polymer enzyme reactors have become a major area of research interest, from their fundamental aspects to applications in bio-living science. However, the polymer materials, that enable the controllable enzymolysis based on ultraviolet-visible (UV)-responsive properties, have remained unexplored. Herein, an enzyme reactor was fabricated by immobilization of l-asparaginase on an UV-responsive porous polymer membrane (UV-PPMER), which consisted of poly(styrene-maleicanhydride-4-[(4-methacryloyloxy)phenylazo]benzoic acid) [P(St-MAn-MPABA)], and explored its controllable enzymolysis. By controlling the “on/off” switch of 365 nm UV irradiation, the configuration of polymer membrane surface changed to improve and tune the enzymolysis. Using l-asparagine (L-Asn) as the substrate, the enzymatic efficiency of the UV-PPMER was evaluated by a chiral capillary electrophoresis technique. Upon UV irradiation, the PMPABA moiety in the membrane changed from a trans- to a cisconfiguration and encapsulated the enzyme and substrate into a narrow cavity, further improving the enzymatic efficiency due to the confinement effect. It was found that the enzymatic reaction rate with the UV-PPMER under UV irradiation (13.3 mM min−1) was 4.5 times higher than that of UV irradiation was off (2.94 mM min−1). Additionally, the low cytotoxicity and excellent UV-responsivity of UV-PPMER were verified in the living cells and serum samples.

Immobilization of l-asparaginase on gold nanoparticles for novel drug delivery approach as anti-cancer agent against human breast carcinoma cells

l-asparaginase is an amidohydrolase enzyme that is widely identified as one of the most potential anti cancerous drugs. Nevertheless, this drug is poorly bioavailable and hence its pharmaceutical uses are limited. To improve its bioactivity, l-asparaginase was loaded on gold nanoparticles (GNPs) along with Arg-Gly-Asp (RGD) peptide that direct the enzyme to targeted cancer cells with aim to enhancing the anticancer efficiency. Successful preparation of GNPs and the conjugate (GNPs-PEG-l-asparaginase-RGD) was verified and delineated by employing UV–VIS spectrophotometer, FTIR, XRD, FE-SEM, and TEM. UV–VIS spectrophotometer, Fourier Transform Infrared (FT-IR), X-Ray Diffraction (XRD), Field Emission Scanning Electron Microscopic (FE-SEM), and Transmission Electron Microscopy (TEM). The efficiency of the GNPs and conjugate to target and distribute in MCF-7 cells was evaluated using high fluorescent signals and confirmed by fluorescence microscopy. A variety of parameters were tested to investigate the anticancer potential of each compound toward MCF-7 cells in vitro. The conjugate demonstrated significant antioxidant effects with high tumor targeting efficacy and distribution in MCF-7 cells. It caused a decrease in cell proliferation rate and clonogenicity of MCF-7 cells, while initiating apoptosis and promoting cell cycle arrest at the G2/M, as confirmed by flow cytometry analysis. These effects were coupled with upregulating the pro-apoptotic p53 while downregulating the anti-apoptotic Bcl-2, which resulted in an alleviation of the mitochondrial membrane potential (MMP) and, thereby, the secretion of cytochrome c. The outcomes of the present study propose the high feasibility for further development of the conjugate as a novel agent against cancer cells.

Immobilization of L-Asparaginase on Magnetic Nanoparticles for Cancer Treatment.

In this presented work, magnetic poly(HEMA-GMA) nanoparticles were synthesized, characterized, and used for immobilization of an anti-leukemic enzyme L-asparaginase. The average particle size of the synthesized magnetic nanoparticles was found to be as 117.5nm. L-asparaginase was successfully immobilized onto the synthesized magnetic nanoparticles, and attached amount of L-asparaginase was found to be as 66.43mg/g nanoparticle. The effects of the medium pH, temperature, and substrate concentration on the L-asparaginase activity were also tested. Optimum pH of the free and immobilized L-asparaginase was found to be as 7.5 and 6.5, respectively. Optimum temperature of the free L-asparaginase was 45°C, while optimum temperature was shifted to 55°C after immobilization onto the magnetic nanoparticles. Also, kcat value of free L-asparaginase (47,356min-1) was calculated to be higher than that of immobilized L-asparaginase (497min-1). Thermal stability of both asparaginase preparation was followed for 10h, and at the end of the incubation time, free asparaginase almost lost its activity, while immobilized asparaginase protected 50% of its initial activity. Storage stabilities of free and immobilized asparaginase were also tested, and at the end of the 40days storage, free asparaginase lost all of its activity, while immobilized asparaginase still showed 30% activity. Operational stability of immobilized asparaginase was tested for 8 successive usage, and immobilized asparaginase lost only 15% of its initial activity. In present study, activities of free and immobilized L-asparaginase were tested in artificial human serum medium, to foresee the in vivo efficiency, and it was demonstrated here that immobilized L-asparaginase protected its 74.74% of its initial activity in artificial serum medium.

Development and characterization of a novel l-asparaginase/MWCNT nanobioconjugate.

The enzyme l-asparaginase (ASNase) presents effective antineoplastic properties used for acute lymphoblastic leukemia treatment besides their potential use in the food sector to decrease the acrylamide formation. Considering their applications, the improvement of this enzyme's properties by efficient immobilization techniques is in high demand. Carbon nanotubes are promising enzyme immobilization supports, since these materials have increased surface area and effective capacity for enzyme loading. Accordingly, in this study, multi-walled carbon nanotubes (MWCNTs) were explored as novel supports for ASNase immobilization by a simple adsorption method. The effect of pH and contact time of immobilization, as well as the ASNase to nanoparticles mass ratio, were optimized according to the enzyme immobilization yield and relative recovered activity. The enzyme–MWCNTs bioconjugation was confirmed by thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FTIR), Raman and transmission electron microscopy (TEM) studies. MWCNTs have a high ASNase loading capacity, with a maximum immobilization yield of 90%. The adsorbed ASNase retains 90% of the initial enzyme activity at the optimized conditions (pH 8.0, 60 min, and 1.5 × 10−3 g mL−1 of ASNase). According to these results, ASNase immobilized onto MWCNTs can find improved applications in several areas, namely biosensors, medicine and food industry.

Immobilization of l-asparaginase on aspartic acid functionalized graphene oxide nanosheet: Enzyme kinetics and stability studies

There is an increasing interest in using l-asparaginase in medical fields and food processing industries. Enzyme immobilization is an attractive field to improve l-asparaginase activity and stability. Graphene oxide (GO) is a promising candidate for enzyme immobilization due to its large specific surface area. In this study, GO was first functionalized with l-aspartic acid (GO-Asp), and then l-asparaginase was immobilized on the GO-Asp either physically or through chemical conjugation. A significant enzyme loading was achieved through covalent immobilization (100% immobilization efficiency). Stability of free and the immobilized l-asparaginase was examined at various temperatures (20–60 °C) and pH (5–9). The covalently immobilized l-asparaginase showed higher enzyme activity than free enzyme at pH 8 with the maximum recovered activity of 100%, 90.5% and 40.6% after 24 h of incubation at 20 °C, 40 °C and 60 °C, respectively. In addition, the covalently immobilized l-asparaginase on GO-Asp showed 42% recovered activity after eight continuous reaction cycles at 60 °C. The kinetic parameters of the immobilized and free enzyme were also calculated, indicating no significant changes in the enzyme affinity through covalent conjugation. The results clearly reflect the suitability of GO-Asp as a nanosheet support for l-asparaginase loading as well as its usage in future industrial applications.

Recent research progress on microbial L-asparaginases.

L-Asparaginases (EC 3.5.1.1) are enzymes that catalyze the hydrolysis of L-asparagine to L-aspartic acid and found in a variety of organisms from microorganisms to mammals. However, they are mainly expressed and produced by microorganisms. Microbial L-asparaginases have received sustained attention due to their irreplaceable role in the therapy of acute lymphoblastic leukemia and for their inhibition of acrylamide formation during food processing. In this article, we review the application of microbial L-asparaginases in medical treatments and acrylamide mitigation. In addition, we describe in detail recent advances in the existing sources, purification, production, properties, molecular modification, and immobilization of L-asparaginase.

L-Asparaginase: A Promising Chemotherapeutic Agent

ABSTRACTThis article comprises detailed information about L-asparaginase, encompassing topics such as microbial and plant sources of L-asparaginase, treatment with L-asparaginase, mechanism of action of L-asparaginase, production, purification, properties, expression and characteristics of l-asparaginase along with information about studies on the structure of L-asparaginase. Although L-asparaginase has been reviewed by , our effort has been to include recent and updated information about the enzyme covering new aspects such as structural modification and immobilization of L-asparaginase, recombinant L-asparaginase, resistance to L-asparaginase, methods of assay of L-asparagine and L-asparaginase activity using the biosensor approach, L-asparaginase activity in soil and the factors affecting it. Also, side-effects of L-asparaginase treatment in acute lymphoblastic leukemia (ALL) have been discussed in the current review. L-asparaginase has been and is still one of the most widely studied therapeutic enzymes by researchers and scientists worldwide.

Immobilization of l-asparaginase on the microparticles of the natural silk sericin protein and its characters

The natural silk sericin recovered from Bombyx mori silk waste by the degumming processing in the high-temperature and high-pressure is a macromolecular protein. Amino acid composition and molecular weight range of the sericin protein as a vector for enzyme immobilization were investigated. The silk sericin protein with different molecular mass from 50 to 200 kDa was poorly soluble microparticles with an average size of about 10 μm. Anti-leukemic enzyme l-asparaginase ( l-ASNase) was covalently conjugated on the microparticles of the sericin protein. The immobilized l-ASNase on the natural support by cross-linking with glutaraldehyde maintained 62.5% of the original activity of the enzyme. The K m of sericin-conjugates was 8 times lower than that of native l-ASNase. The bioconjugation of l-ASNase widened the optimum reactive temperature range of the enzyme. The immobilized l-ASNase showed significantly higher stability when the temperature raised to 40–50°C, it also showed preferable resistance to trypsin digestion as compared with native enzyme. The results are discussed regarding the possible explanations of sericin-induced enzyme stability, as well as the possible applications of immobilized l-ASNase research.

Immobilization of L-asparaginase into a biocompatible poly(ethylene glycol)-albumin hydrogel: I: Preparation and in vitro characterization.

The feasibility of the immobilization of Escherichia coli L-asparaginase into a hydrogel matrix made of poly-(ethylene glycol) (PEG) and BSA was demonstrated. After immobilization a 200-fold increase in the Km value was observed. The use of an L-aspartic acid analogue, carbobenzoxy-L-aspartic acid and surface modification by methoxy-PEG of molecular mass 5 kDa cause a only a slight gain in affinity of the enzyme for its natural substrate. The immobilized L-asparaginase has an optimal activity over a larger range of pH than the native enzyme, owing to the effect of the matrix. At a physiological pH of 7.3, the immobilized enzyme retained 90% of its activity compared with only 43% for the native form. The immobilized enzyme retained a high proportion of its initial activity, more than 90% after 50 days of incubation at 37 degrees C, even in the presence of its substrate. This may be compared with a half-life of 2 days observed for native enzyme incubated under the same conditions. These results suggest that the BSA-PEG matrix can be very useful for enzyme immobilization and, taking into account the good biocompatibility of the matrix, one can expect that this matrix will provide a functional bioreactor for use in vivo.

The immobilization of l-asparaginase on porous hollow fiber plasma filters

The enzyme l-asparaginase has been used as a drug to treat acute lymphocytic leukemia. However, its effectiveness is limited as a free drug and it is more useful when immobilized. In this study, l-asparaginase has been immobilized onto the porous polypropylene (PP) hollow fibers of a plasma filter. The fibers were first radiation grafted with polymethacrylic acid (poly(MAAc)) which introduced carboxyl groups onto their surface, and within their pores. l-asparaginase was then covalently bound to the fibers using carbodiimide and N-hydroxy succinimide (NHS). In vitro tests were performed on the plasma filters to determine the immobilized enzyme's stability over time, K m , pH optimum and activity dependence on flow rate. In vivo tests have also been carried out using a sheep as an animal model. We concluded that plasmapheresis using an immobilized l-asparaginase plasma filter could become a useful clinical tool for the treatment of acute lymphocytic leukemia.