Introduction

Asparaginase is used in the production of food and as a medicine. l-asparaginase is a drug that is employed to cure acute lymphoblastic leukemia. This is injected into a nerve, tendon, or under the skin (Monajati et al. 2018). There is also a pegylated variant. It is used in the food industry to reduce acrylamide levels. The first finding and development of asparaginase as an anti-cancer drug in 1953 followed the scientific observation that lymphomas had returned in rats and mice after guinea pig serum therapy (Zadeh Hosseingholi et al. 2020). Later, the asparaginase enzyme was shown to be responsible for suppressing the tumor, not the serum. Since comparing various asparaginases, researchers discovered that the one extracted from Escherichia coli and Erwinia chrysanthemi had better anti-cancer properties. E. coli has since become the primary source of asparaginase due to its ease of production in vast quantities (Qeshmi et al. 2018). Figure 1 shows that l-asparaginase hydrolyzes asparagine to aspartic acid. Thus, preventing the feeding of asparagine to tumor cells and the formation of acrylamide in heat-processed foods (Qeshmi et al. 2018).

Fig. 1
figure 1

l-asparaginase hydrolyzes asparagine to aspartic acid, thus preventing feeding of asparagine to tumor cells and formation of acrylamide in heat-processed foods. Reprinted from Qeshmi et al. (2018) with permission from Elsevier. License Number: 5121780717424

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Common side effects are allergies, pancreatitis, blood clotting issues, elevated blood sugar, kidney complications, and liver disease. Asparaginase works by breaking down the amino asparagine needed to generate protein in cancer cells. Coagulopathy can also be caused by inhibiting protein syntheses such as coagulation and anticoagulant factors (Avramis et al. 2002). Bone marrow regression is normal, but it is usually minor to modest, infrequently exceeds medical importance, and treatment is seldom needed. Pancreatitis is another common side effect. These side effects are due to asparaginase’s dual activity, as it can also hydrolyze L-Glutamine to Glutamic acid and ammonia (Kante et al. 2019).

In the United States, asparaginase was sanctioned for medicinal usage in 1978. It is classified as an essential medicine by the World Health Organization. Escherichia coli or Erwinia chrysanthemi are often used to produce asparaginase at a large scale (Kim et al. 2016). Asparaginases have a variety of commercial and medicinal applications. Medical asparaginase is mainly derived from E. coli strains. In 1998, branded formulations such as asparaginase Medac, Ciderolase, and Oncaspar were available (all with various chemical and pharmacological properties) (Avramis et al. 2002). Spectrila is innovative recombinant asparaginase from E. coli. Dickeya dadantii (formerly Erwinia chrysanthemi) asparaginase is now branded as crisantaspase and is sold in the United Kingdom under the brand name Erwinase (Brayfield 2017). Some mast cell tumor protocols use one of the E. coli asparaginases sold under the brand name Elspar to treat acute lymphoblastic leukemia. Unlike most other chemotherapy drugs, asparaginase can be administered intramuscularly, subcutaneously, or intravenously without causing tissue inflammation (Appel et al. 2007).

Asparaginase is based on the circumstance that acute lymphoblastic leukemia cells and other suspected tumor cells cannot synthesize the non-essential amino acid asparagine. Therefore, leukemic cells need a large amount of asparagine (Fernandes et al. 2017). In addition, these leukemic cells rely on asparagine in the bloodstream to survive. Asparaginase converts asparagine into aspartic acid and ammonia, which results in the unavailability of this non-essential amino acid to leukemic cells, leading to their death (Wlodarczyk et al. 2019).

Asparaginases are most often used in different sectors, such as sensing, food and pharmaceutical (Fig. 2) (Chand et al. 2020; Nunes et al. 2020). Asparaginases act as food handling support that helps to prevent the development of acrylamide, a potential cancer-causing agent, in starchy foods like cookies, biscuits, and fried potatoes (Qeshmi et al. 2018). Acrylaway and PreventASe are two brand names for asparaginases. Acrylamide is commonly produced as starchy foods are cooked. Figure 3 shows the use of asparaginases in the food industry to reduce the acrylamide (da Cunha et al. 2019). The amino acid asparagine, which is naturally found in starchy foods, undergoes a mechanism known as the Maillard reaction when heated and is liable for the brown color, crust, and toasted taste of baked or fried foods (Xu et al. 2016). The Maillard reaction also produces suspected carcinogens including acrylamide and certain heterocyclic amines. Asparaginase before baking or cooking food is converted into another common amino acid, aspartic acid, and ammonium (Cachumba et al. 2016). Asparagine does not then participate in the Maillard reaction, which leads to a significant reduction in acrylamide formation. Because of other pathways of shaping small asparagine independent, complete isolation of acrylamide is improbable. As an aid for food production, asparaginases can effectively reduce acrylamide levels in a range of starchy foods by up to 90% without altering the taste or appearance of the finished product (Muneer et al. 2020).

Fig. 2
figure 2

Strategies for l-asparaginase confinement and its potential applications in sensing, food and pharmaceutical sectors. Reprinted from Nunes et al. (2020) with permission under the terms and conditions of the creative commons attribution (CC BY) license

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Fig. 3
figure 3

asparaginase role in food industry. Reprinted from da Cunha et al. (2019) with permission from Elsevier. License Number: 5121780884450

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Herein, we reviewed recent progresses related to the useful exploitation of a several nanostructured materials, as carrier matrices, for asparaginases immobilization. The work also emphases the catalytic potentials and applications of immobilized asparaginases-based nano-biocatalytic systems in the medical, nutritional, and biotechnological sectors (Fig. 4).

Fig. 4
figure 4

Potential applications of asparaginases

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Enzyme immobilization

Despite the many benefits of using enzymes over conventional catalysts, there are a few functional issues regarding their usage in industrial applications. Enzymes are inherently expensive, but the expense of isolating and purifying them is much higher than the cost of ordinary catalysts (Zdarta et al. 2018; Al-Maqdi et al. 2021). When isolated from their natural habitats, they are especially susceptible to different denaturing conditions due to their protein composition. Their exposure to process conditions like temperature, pH, and trace amounts of substances will serve as inhibitors, increasing their costs (Homaei et al. 2013; Bilal and Iqbal 2020; Bilal et al. 2021a). In contrast to traditional heterogeneous chemical catalysts, most enzymes in homogeneous catalysis systems are dissolved in water, resulting in substance contamination and preventing their recovery in the active state for reuse from most reaction mixtures (Liang et al. 2020).

The use of an immobilization technique is one of the most common approaches suggested to address these limitations. Immobilization is a scientific term for the mechanism of attaching enzymes to or inside stable supports, resulting in a heterogeneous immobilized enzyme framework (Khoshnevisan et al. 2019; Bilal et al. 2020; Hussain et al. 2021). Most enzymes are bound to the cellular cytoskeleton, membrane, and organelle structures in their immobilized state, which mimics their normal living cell mode. Strong support structures usually stabilize the enzymes’ configuration, and their functions are maintained as a result (Husain 2017). In this way, real-time enzymes are more stable and resilient to environmental changes than free solution enzymes (Wang et al. 2020). In addition, heterogeneous immobilized enzymes systems enable quick recovery of both enzymes and ingredients, a wider variety of bio-reactor designs, continuous operation of enzymatic processes, and rapid reaction endings (Bernal et al. 2018). In contrast to free enzymes, the immobilized biocatalysts present low activity and high apparent Michaelis constants because of difficult accessibility to the substrate (Kress et al. 2002).

In recent years, there has been a lot of curiosity and focus on the ability of immobilized enzymes (Bommarius and Riebel-Bommarius 2004). Immobilized enzymes usually are more stable and more straightforward to manage than their free versions. In proteases, the rate can be considerably slowed down while they remain immobilized, and reaction products may not be contaminated with the enzyme (particularly advantageous in the food and pharmaceutical industries) (Massolini and Calleri 2005). These modifications are the result of fundamental fluctuations made into the enzyme particle by the immobilization process used and the formation of a microenvironment for the enzyme to function in that is distinct from the bulk solution (Rodrigues et al. 2013). As a result, the substance will be clean, free of all environmental contaminants, and simple to separate from the solution. Enzymes can be immobilized using several techniques and divided into two categories: physical, which involves feeble connections among the carrier and the enzyme, and chemical, which comprises the establishment of covalent connections among the carrier and the enzyme (de Andrades et al. 2019).

In several states, enzymes may help accelerate reactions, including successfully processed molecules, aggregates with other species, or surface molecules. Many seeking to manipulate them for scientific purposes have been particularly interested in the attached or “immobilized” condition (Brena et al. 2013). In certain circumstances, the use of immobilized catalysts has vastly increased both the technological and economic efficiency of manufacturing processes. When incapacitating Aspergillus oryzae aminoacylase to solve synthetic racemic d–l amino acids in 1966, Chibata and coworkers revealed the first commercial use of immobilized enzymes (Liu and Dong 2020). In the industrial production of carbohydrates, amino acids, and pharmacology, immobilized enzymes are often included (Yushkova et al. 2019). In some production processes, entire microbial cells carrying the target enzyme are incapacitated as catalysts (Basso and Serban 2019).

Apart from their use in manufacturing applications, immobilization methods are used to create various biotechnology devices such as diagnostics, bioaffinity chromatography, and biosensors (Nguyen et al. 2019). Therapeutic uses, like the usage of enzymes in extracorporeal diversions, are also anticipated (Fernandez-Lopez et al. 2017). Immobilization technology has advanced exponentially over the last few decades and has gradually turned into a coherent project. However, there is still room for improvement (Al-Qodah et al. 2017). Extending the usage of restrained enzymes to other functional practices will necessitate the creation of new methodologies and a better understanding of the ones currently in use (Bilal and Iqbal 2019a; Bilal et al. 2021b).

In 1916, scientists made the first experimental finding that contributed to discovering immobilized enzymes (Gomes-Ruffi et al. 2012). Invertase was shown to have the same activity when ingested on a solid at the bottom of the reaction tank, such as charcoal or aluminum hydroxide, as when evenly dispersed throughout the solution. This discovery led to the creation of today’s enzyme immobilization techniques (Homaei et al. 2013).

In comparison to usable surface areas, early immobilization strategies produced very low enzyme loadings. Numerous covalent approaches of enzyme arrest were established in the 1950s and 1960s (Hartmann 2005). Since the 1960s, > 5000 papers and official documents on enzyme halt procedures have been issued. Quite a lot of enzymes have been incapacitated in various ways, and > 1 million restrained enzymes have been employed as a catalytic agent in several extensive progressions, such as penicillin G acylase, invertase, lipases, proteases, and so on (Kallenberg et al. 2005). Although enzyme immobilization has been considered for a long time, the publication of current investigation and appraisal credentials shows that there is still a lot of interest in this field.

Immobilized enzymes have proven to be highly useful for industrial applications in many ways. They have several benefits above enzymes in solution, comprising cost, constancy, and the ability to be quickly extracted from the solution for wholesome product separation (Sigurdardóttir et al. 2018). In addition, attaching an enzyme to a stable help will improve its resilience to environmental changes, including pH and temperature (Boudrant et al. 2020). Five common ways to immobilize enzymes are adsorption, entrapment, encapsulation, covalent binding, and cross-linking or bioconjugation (Nguyen and Kim 2017; Bilal et al. 2018). The binding of weak or covalent bonds is possible with physical or chemical assistance. However, physical attachment is generally poor, and the enzyme bound to a carrier can scarcely maintain under industrial conditions. A synthetic resin may be used as reinforcement, an inorganic polymer such as zeolite, silica, or a biopolymer (Bilal et al. 2019). An enzyme is entangled in an organic polymer (gel grid) or silica gel network or a membrane unit such as hollow fibres or a microcapping enzyme (Zdarta et al. 2018). The synthesis of the polymeric network in possession of the enzyme is required for entrapment. The final category consists of using a dual functionality to build carrier-free macro-particles by cross-link enzyme aggregates or crystals (Gonçalves et al. 2019). However, no one tool or support is appropriate for all enzymes and applications (Chapman et al. 2018). This is due to the wide range of chemical properties and compositions of enzymes, substratum and materials’ diverse qualities, and the product’s numerous applications. However, any of the approaches can have benefits and disadvantages (Sigurdardóttir et al. 2018). Adsorption is convenient, inexpensive, and efficient, but it is often reversible; covalent connection and cross-linking are adequate and stable. However, they are costly and can quickly degrade enzymes. The membrane reactors have intrinsic diffusional problem confinement, entrapment, and micro-encapsulations (Nguyen and Kim 2017).

Immobilization support materials

When it comes to determining the efficacy of the immobilized enzyme mechanism, the matrix properties are important. Despite the fact that solid support immobilization is a well-established technology, there are no universal standards for selecting the best support for specific usage. According to their chemical structure, supports may be classified as inorganic or organic. Natural and synthetic polymers are two kinds of organic supports (Brena et al. 2013). The physicochemical characteristics of the matrices (such as mean particle diameter, swelling activity, mechanical power, and tensile behavior) are crucial for the immobilized systems’ efficiency and also specify the type of reactor used under technical conditions (i.e., stirred tank, fluidized, or fixed beds) (Soares et al. 2011). Essential features of materials that could be used as support matrices for immobilization as shown in Fig. 5. In particular, pore parameters and particle size decide the overall surface area and greatly influence the enzyme’s ability to bind. Non-porous media have a reduced charge potential and diffusion restrictions. Consequently, the porous support is commonly supported, because the wide surface area of the enzyme enhances the loading of the immobilized enzymes (Reichardt et al. 2018). In addition, a balanced pore distribution is highly requisite to maximize the enzyme loading and flow properties. In most industrial applications, organic matrices are used despite the many advantages of inorganic carriers (like their strong tolerance with physical, chemical, and microbial degradation). The degree of action of an immobilized enzyme is defined most notably by its hydrophilic nature (Ke et al. 2018).

Fig. 5
figure 5

Essential features of support materials to be taken into account for designing an immobilization approach

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Agarose is a robust matrix that has a long history of use. Additionally, agarose’s hydrophilic nature, ease of derivatization, absence of charged groups (which prevents nonspecific adsorption of substrate and products), and commercial availability all contribute to its advantages (Bilal et al. 2018). However, the high expense of agarose and other brittle materials is a significant drawback. One way to circumvent this issue is to employ reversible immobilization techniques that allow for matrix regeneration and reuse. For example, macroporous acrylic polymers such as Eupergit® C and Sepabeads® EC, which includes Eupergit® C, are the most effective carriers of covalent enzyme immobilization in industrial applications (Almulaiky et al. 2019).

Nanomaterials may be advantageous as support materials for enzyme immobilization. They possess the ideal properties for balancing the three critical parameters that define biocatalyst efficiency: density, mass transfer resistance, and effective enzyme packaging (Zhong et al. 2020). Nanotechnology has generated many new possibilities for immobilizing enzymes in biotechnology, immunosensing, and biomedicine (Sharifi et al. 2020). Recently, nanoscale immobilization enzymes such as silicone microspheres, fibers, tubes, and numerous metal and magnetic nanoparticles have been discovered (Bilal and Iqbal 2019b).

Nanomaterials

Nanoparticles, nanotubes, mesoporous materials, and fibrous nano-membranes have been studied in many ways using nanotechnology to immobilize enzymes on the surface of nanoparticles (Cipolatti et al. 2016). As a result, much has been said about the use of nanomaterials and their benefits in interactions with biocatalysts. Since nanomaterials are not porous, the surface of nanoparticles contained all enzyme molecules. Internal limitations of diffusion are thereby avoided (Sheldon and Pelt 2013). If the enzyme is correctly placed on the support surface, enzymes immobilized on non-porous nanomaterials can be multipoint attached to the support to increase its stability and still operate in very large or even insoluble materials; this is not true for conventional porous supports (Barbosa et al. 2013). However, the immobilization of the support’s exterior surface poses certain issues that must be addressed. The enzyme is no longer shielded from hydrophobic interfaces such as gas bubbles. The enzyme molecules in one particle can interact with the enzyme molecules in another particle (allowing proteolysis) (Betancor et al. 2005). This problem can be addressed by coating the immobilized support with a polymer that prevents these harmful contacts, preventing enzyme inactivation in stirred systems (Betancor et al. 2004). Diffusion barriers reduce enzyme activity or even stability. When nanoparticles are used, this result is not feasible. Diffusion complications can also be beneficial in the case of coimmobilized enzymes. Owing to these gradients, the second enzyme, when coimmobilized, works on high concentrations of the first enzyme’s output, and the apparent activity of the combi-enzyme catalyst can be greatly enhanced in some situations (Rodrigues et al. 2013). For example, in a three-enzyme system, this allowed for even more effective NADH recycling, surpassing even the use of equal quantities of free enzymes. Non-porous nanomaterials, once again, are unable to produce this effect. As a result, nanomaterials have several advantages; however, in some situations, the deficiency of some of the possessions of immobilization on typical porous supports could result in the absence of some desirable effects (Cipolatti et al. 2016).

Nanoparticles normally have a diameter of 1–100 nm and are made up of hundreds of atoms, but in some instances, such as those formed by miniemulsion, they may be as large as 500 nm. Clusters of particles are described as particles with a diameter less than 1 nm. Nanoparticles with a diameter of less than 10 nm are interesting, because all atoms being on or near the surface (Shi et al. 2015). Nanostructures have been used to promote enzyme immobilization via various mechanisms, including enzyme adsorption, covalent binding, enzyme encapsulation, and refined process combinations (Yang et al. 2016). It should be noted that controlling enzyme–support interactions is critical for understanding the various implementations and improvements that can be made and controlling the alignment of the enzyme can be particularly important (large substrates, immobilization area) (Barbosa et al. 2013). Wide or insoluble substrates, for example, necessitate the use of nanoparticles as a help, as do situations where immobilized enzymes cannot be used on porous supports (Barbosa et al. 2014).

Nanomaterials used for asparaginase immobilization

Magnetic nanoparticles

One of the most commonly used solids in immobilization studies is nanostructured metals which have a wide surface area for the immobilization of the enzyme. Magnetic nanoparticles have attracted much attention among nanostructured materials because of their peculiar physical and mechanical features, such as super-paramagnetism, high porosity and high viscosity. Furthermore, in the presence of an external magnetic field, even magnetic nanoparticles exhibit basic separation properties (Vaghari et al. 2016). Moreover, the ability of magnet nanoparticles to modulate them with a polymer and keep substituents on their surfaces in several applications in the last years has become commonplace (Orhan et al. 2019). Immobilization of asparaginase on magnetic nanoparticles is schematically shown in Fig. 6 (Ates et al. 2018).

Fig. 6
figure 6

Immobilization of l-ASNase onto the magnetic Fe3O4–chitosan core–shell particles. Reprinted from Ates et al. (2018) with permission from The Royal Society of Chemistry. This Open Access Article is licensed under a Creative Commons Attribution-Non-Commercial 3.0 Unported Licence

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Orhan and Uygun synthesized the anti-Leukemia-l-asparaginase enzyme, which is characterized using magnetic poly(HEMA-GMA) nanoparticles (2020). The magnetic nanoparticles were calculated to have a mean particle size of 117.5 nm. The synthesized magnet nanoparticles with a total of 66.43 mg/g nanoparticles were successfully immobilized with l-asparaginase. l-asparaginase activity was also measured concerning the medium pH, temperature, and substrate concentration. At their optimal concentrations, free and immobilized l-asparaginase had pH values of 7.5 and 6.5, respectively. The optimal temperature for free l-asparaginase was 45 °C, but after immobilization on magnetic nanoparticles, the optimal temperature was shifted to 55 °C. Additionally, Kcat was determined to be greater than the value of incapacitated l-asparaginase in Free l-asparaginase. The thermal stability of the two asparaginase preparations was monitored over 10 h. At the end of the period, free asparaginase almost completely lost activity. In contrast, immobilized asparaginase retained 50% of its initial activity. The storage stabilities of free and incapacitated asparaginase were investigated, and after 40 days of storage, free asparaginase lost its whole operation, while incapacitated asparaginase retained 30% of its activities. Eight successive uses of rendered immobile asparaginase were evaluated, and only 15% of the original activity was lost. To predict in vivo development, free and incapacitated l-asparaginase behaviors were determined in an artificial human serum medium. It was discovered that rendered immobile l-asparaginase covered 74.74% of its initial function. Noma et al. (2020) prepared amino (Fe3O4/SiO2/NH2) and carboxylic-functional (Fe3O4/SiO2/COOH) particles. Compared to free enzymes, immobilized enzymes were more robust over a wide variety of temperatures and pH values.

Gold nanoparticles

Gold is often worth noting as support for biocatalyst immobility. Despite the fact that gold nanoparticles are not called practical supports due to cost considerations, they are gaining prominence in catalysis (Venditti et al. 2015). Amylase from Bacillus subtilis, xylanase from Thermomyces lanuginosus, peroxidase from Porphyromyces chrysosporium, cellulase from Trichoderma reesei, and superoxide dismutase (bovine) are only a few examples of enzymes incapacitated on gold nanoparticles (Cipolatti et al. 2016). According to Yan et al. (2012), the following characteristics distinguish gold nanoparticles (GGNs) from other nanoparticles: it has a porous network, transparent, bicontinuous structure that promotes strong adsorption and can accommodate high enzyme loads; (ii) the structural unit is modifiable in a wide variety of configurations and functions ranging from several nanometers to several microns; (iii) the structural unit is tunable in a wide range of dimensions ranging from a few nanometers to several microns, which accommodates a wide variety of enzyme molecule (v) NPG is generated without the use of organic or surfactants, resulting in exceptionally clean surfaces that are free of toxic molecules or ions that could interfere with enzymes.

Due to their basic properties, such as chemical resistance, enzyme tolerance, and low cytotoxicity, gold nanoparticles (GNPs) are an excellent delivery mechanism for various anti-cancer drugs. Additionally, GNPs are biocompatible and easy to conjugate with other biologically active compounds, such as antibodies and enzymes. Additionally, their optical properties have unique properties that allow them to rapidly bind to active groups such as amine and thiol and thus modify the cell surface (Safary et al. 2019). Due to its ability to join cells through the endocytosis mechanism, the RGD sequence (GRGDSP) is a favored candidate for the majority of cancer therapies (Sotnikov et al. 2019). In endocytosis, hydrophilic agents are cell-penetrating peptides (CPPs) that facilitate receptor binding to the cell membrane, resulting in successful internalization. The RGD series is a fibronectin derivative (Hamdan et al. 2019). A protein that performs a variety of functions in the extracellular matrix, including assisting cell development. Thus, cells that have clustered adhesion points in the extracellular matrix may be used to identify RGD. This may result in malignant cells overexpressing the RGD receptor that are particularly susceptible to certain CPPs (Mabonga and Kappo 2020).

Al-Dulimi et al. (2020) enhanced the bioactivity of l-asparaginase by loading it onto gold nanoparticles (GNPs) along with the Arg-Gly-Asp (RGD) peptide (Fig. 7). These methods seek to boost anti-cancer efficiency by targeting individual cancer cells with the enzyme. Fluorescence microscopy was used to test and verify the GNPs and conjugate’s ability to target and propagate in MCF-7 cells. Every compound was evaluated in vitro for its anti-cancer activity against MCF-7 cells using a variety of parameters. The conjugate demonstrated significant antioxidant activity in MCF-7 cells and high tumor-targeting efficacy and dissemination. According to flow cytometry analysis, it inhibited cell proliferation and clonogenicity in MCF-7 cells, thus inducing apoptosis and promoting cell cycle arrest at the G2/M phase. These findings were associated with an increase in pro-apoptotic p53 expression and a decrease in anti-apoptotic Bcl-2 expression, resulting in reduced mitochondrial membrane potential (MMP) and, therefore, cytochrome c secretion. According to the results of this study, the conjugate has a strong potential for further development as a novel cancer-fighting agent.

Fig. 7
figure 7

Schematic illustration of the formation of the GNPsePEG-RGD-asparaginase (conjugate) and cellular uptake. Reprinted from Al-Dulimi et al. (2020); this is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

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Graphene oxide

One of the most commonly suggested nanomaterials is graphene oxide (GO) (Liu et al. 2013). GO recently caught a great deal of interest in several areas, including nanoelectronic devices, nanocomposed materials, solar cells, biosensors and biomedicine because of its unusual composition and different electric, thermal and mechanical properties (Singh et al. 2018). GO nanosheets can be used in transmission lines, particularly in biomedicine because of their double faces and ends, fitted with reactive functional groups such as hydroxyl, epoxy, and carboxylic acid (Dreyer et al. 2014). It is useful to immobilize various substances, including enzymes, medicines, chromosomes, and molecules of fluorescence (Heidarizadeh et al. 2017). However, it was challenging to use in the biomedical field due to GO toxicity and poor dispersion stability in biological media. On the other hand, the surface functionalization with GO makes it more biocompatible and effective biomedical system (Shim et al. 2016). Moreover, GO functioning as an aid for immobilization of enzymes improves the capacity for loading, stability, and reusability of enzymes, by reducing structural damage caused by high GO interfacial tension (Mehnati-Najafabadi et al. 2018). For asparaginase immobilization, Monajati et al. (2018) used GO-Asp nanocomposite (GO-Asp-CDI/Enz) for covalent immobilization of l-asparaginase (Fig. 8). l-Aspartic acid (GO-Asp) was used to functionalize GO, and then l-asparaginase was immobilized on the GO-Asp either physically or chemically. Covalent immobilization was used to obtain a significant enzyme loading. The immobilized and free enzymes show high kinetic parameters that prove that covalent conjugation has no considerable impact on enzyme affinity. The results show the appropriateness and scope for use in future industrial applications of GO-Asp as nanosheet support for l-asparaginase loading.

Fig. 8
figure 8

Reproduced from Monajati et al. (2018) with permission from Elsevier. License Number: 5121781311571

Immobilization of asparaginase on graphene oxide.

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Synthetic nanomotors

Synthetic micro/nanomotors have attracted a lot of interest for various biomedical and environmental applications (Li et al. 2017). Efficient motion can be achieved by converting a locally available fuel into force and acceleration or using an external input, such as magnetic or ultrasound energies (Li et al. 2016). Chemical combustibles such as hydrogen peroxide can restrict in vivo the use of catalytic engines. Nanowire engines are driven acoustically by continuous asymmetric streaming, producing a finite propulsion speed perpendicular to the oscillation trajectory and along the symmetry axis of the device (Kim et al. 2015). Targeted distribution, precision medicine, biological target sensing, detoxification, biological cargo carrying, and collaborative action have all been effective applications of US-propelled engines, which can be used in biological media with high stability (Soto et al. 2016). Such mobile microscale systems may circumvent traditional passive distribution vehicles, which depend on systemic circulation and lack the requisite power and navigation for decentralized delivery and tissue penetration within their passive mass transport restrictions. Rapid internalization and movement inside living cells of gold nanowire motors operated by the US were also used for fast intracellular siRNA transmission for gene silencing. Nobody, however, has yet exploited the enzymatic supply of US-powered micromotors as enzymes to kill cancer cells (Esteban-Fernández de vila et al. 2016).

Uygun et al. (2017) altered ultrasound-propelled nanowires composed of Au/Ni/Au/PEDOT-PPy-COOH segments with asparaginase enzyme and used them as an efficient anti-cancer agent. After immobilization on the nanowire motors’ surface, the enzyme exhibits increased thermal and pH stability, increased tolerance to substrate defense, and increased affinity. The rapid action of the motor-carrying asparaginase contributes significantly to the biocatalytic degradation of asparagine, thus significantly increasing its inhibitory activity against El4 lymphoma cancer cells (92%) in contrast to the free enzyme counterpart (17%) and other control types. Accelerated engine movement increases the enzyme’s association with cancer cells, resulting in enhanced enzyme production in cancer cells. Although current research uses asparaginase and tumor cells EL4 as a model system for inhibiting cancer cells, the same principle may be applied to other forms of enzymes and biomolecules with similar biofunctions.

Carbon nanotubes

Carbon nanotubes (CNTs) have been the subject of extensive research. CNTs are cylinder-shaped graphene sheets with diameters of up to 100 nm. They range in size from mm to 100 nm (Chakravarty et al. 2021). Due to their exceptional structural dimensions, chemical and mechanical properties, strong loading ability, and biocompatibility, both single-walled and multi-walled carbon nanotubes (SWCNT) have been used as carrier materials for enzyme attachment via a range of methods, including direct physical adsorption and covalent attachment (Zhou et al. 2020). For example, when it came to laccase adsorption, carbon nanotubes outperformed other carriers such as silica, spent grain, and coconut fiber by 333, 20, and 714 times, respectively (Cristóvo et al. 2020). Cristóvo et al. (2020) also mentioned that previously no study is carried out to immobilize ASNase on pristine MWCNTs. Therefore, they investigated MWCNTs as a novel physical adsorption immobilization support for ASNase. They also reported that compared to covalent bonding, this physical absorption technique is a low-cost connection process (Mohamad et al. 2015).

Apart from their possible usage in the food industry to prevent the development of acrylamide, the enzyme l-asparaginase (ASNase) possesses significant anti-cancer properties and has been used to treat acute lymphoblastic leukemia. Given their applications, successful immobilization procedures that improve this enzyme’s properties are in high demand (Mohamad et al. 2015). Ulu et al. (2020) prepared and characterized calcium-alginate/multi-walled carbon nanotube hybrid beads (Ca-ALG/MWCNT-COOH) as a novel kind of matrix. According to characterization results, hybridization of ALG and MWCNT-COOH resulted in significant changes in surface morphology and structure. After implementation, the Km value was 1.27 times smaller than the free equivalent, indicating an increase in preference for the substratum. Additionally, despite 14 reuses, the immobilized enzyme retained more than 36% of its initial activity. Additionally, this hybrid material can provide a promising platform for immobilizing commercial enzymes in industries and medical applications.

Silver nanoparticles

SNPs (silver nanoparticles) are considered potential bioactive agents with anti-cancer activity (Baskar et al. 2015). In diabetic wound healing, silver-loaded zirconium phosphate nanoparticles can be used as a bacteriostatic agent. In a mouse model of Dalton’s lymphoma, the ascites tumor showed positive effects in extending the mouse’s life cycle and reducing ascites fluid, resulting in a return to normal body weight (Sriram et al. 2010). Asparaginase, derived from the fungus Aspergillus terreus, has been identified as a promising cancer treatment. Regular cells develop asparagine with the aid of asparagine synthase to maintain normal growth activity. Cancerous cells lack asparagine synthase and must rely on freely circulating asparagine to establish. Asparaginase transforms free asparagine to aspartic acid and ammonia, depriving cancerous cells of asparagine and ultimately leading to cell death (Baskar et al. 2015).

Baskar et al. (2017) used silver nanoparticles to immobilize fungal asparaginase to build a nanobiocomposite. The enzyme activity of an asparaginase-bound silver nanoparticle was found to be higher than that of crude asparaginase. Asparaginase binding to silver nanoparticles is mediated by the primary and secondary amine/amide functional groups. Asparaginase-loaded silver nanobiocomposite was developed with 60–80 nm in size. The cytotoxicity of silver nanobiocomposite of asparaginase was observed to be greater than free asparaginase on the ovarian cancer cell line. Against the ovarian cancer cell line A2780, the silver nanobiocomposite of asparaginase demonstrated greater cytotoxicity than the lung cancer cell line A549. As a result, the asparaginase silver nanobiocomposite can be an important anti-cancer against lung cancer.

Polyaniline nanofibers

Due to its sustainability (high stability at extreme temperatures and pH and microorganism resistance), special conductivity mechanisms (direct electron transfer capacity between an enzyme and a polymer), polyanilines (polymers, nanofibers, and composites) become increasingly important in catalytic processing as energy storage instruments, as sensors and as biological agents. Researchers also showed that they are biocompatible in PC-12 cells (Humpolicek et al. 2012). In reality, leading polymer nanofibers as a stabilization matrix, because of the smaller fibers (indicating a wide area), higher porosity (small diffusion resistance and short distance to the substrate diffusion), and higher conveying capacity and facilitating manufacturing, is recommended over other carriers. Polyaniline-immobilized enzymes have also been used (Zhang et al. 2020). Ghosh et al. (2012) have studied the incapacitated state of enzymes in different concentrations as a novel immobilization aid, and l-asparaginase as a model protein enzyme in their functionality on polyaniline nanofiber surfaces. XPS, XRD, TEM and FTIR have been used to characterize the enzyme and to thoroughly investigate the impact of nanofiber polymer conformations on the enzyme behavior. l-asparaginase was more resistant to denaturation at various temperatures and pH levels than the free enzyme.

Applications of asparaginase

Drug delivery

Nanoparticles have been used as medication administering agents for cancer, HIV, respiratory disorders, and other diseases for decades. Not only can nanoparticles reduce immunogenicity, but they also increase the solubility, half-life, and therapeutic index of medicines (Hubbell and Chilkoti 2012). Numerous delivery systems using nanoparticles have been employed in the past to administer a variety of medicines. Polymer, liposomal, magnetic, platinum, copper, quantum dots, carbon nanotubes, and dendrimers are only a few components included. PEG-l-asparaginase (Oncospar; Enzon) was the first polymeric therapy approved by the US Food and Drug Administration to treat acute lymphocytic leukemia in 1994. (Torchilin 2005). Poly(lactic co-glycolide) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) are two additional polymers used in l-asparaginase-loaded nanoparticles (PHPV). The researchers developed a silk-fiber nanoparticle that enhanced the tolerance and stability of trypsin with the enzyme (Wang and Zhang 2015).

Another method of distribution is using magnetic nanoparticles as an MRI contrast agent. Additionally, the enzyme is immobilized in hydrogel magnetic nanoparticles via a chitosan coating with a layer of hyaluronic acid (Teodor et al. 2009). This enzyme has recently been immobilized in magnetic nanoparticles. Additionally, it incorporates a biocompatible reactive polymer referred to as polymers (2-vinyl-4, 4-dimethylazlactone). After ten uses, it maintains 95.7% of its activity. After 10 weeks of storage, it retains more than 72.6% of its activity (Mu et al. 2014). Additionally, the researcher developed a nanobiocomposite with asparaginase, carbohydrate, methotrexate (Mtx), and fluorescein isothiocyanate (FITC). The FITC imaging property aids in regulating the drug-receptor, while Mtx (a folic acid analog) aids in the distribution of medications (Muthukumar et al. 2014). The enzyme’s non-human nature is one of the significant issues, and antibodies have formed against it. This was accomplished by enclosing it in a dual pore-hollow nanopart that can only move around l-asparagine. The reaction takes place inside the pores, but the antibody and protease are located beyond the hollow nanopores. This prevented the immobilized enzyme from being degraded by the immune system (Ortac et al. 2014).

Antibacterial properties

Industries continue to seek an innovative basis of l-asparaginase with a greater harvest and distinctive properties. This is because its extensive usage generates a high level of marketplace request that is impossible to encounter at the current fabrication rate. According to previous studies, the sources of this enzyme also possess antimicrobial properties. This quality may be used to prevent contamination during the bioprocessing of l-asparaginase (Vimal and Kumar 2017a, b). Both characteristics are also present in the enzyme extracted from these sources that are active against pathogens. Sea origins are more nuanced and less researched for the synthesis of this enzyme. The microorganisms from this basis are anticipated to have unique properties owing to their lenience for a wide variety of environmental conditions (Sivasankar et al. 2013). Amaranthus polygonoides leaves containing l-asparaginase often possess antibacterial and antifungal action against various pathogens, including Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus luteus, Bacillus cereus, Bacillus subtilis, E. coli, Pseudomonas aeruginosa, Klebsiella pneumonia, and Aspergillus niger (Naveena et al. 2012).

Management of cancer

Clementi discovered l-asparaginase for the first time in guinea pig serum in 1922. Broome investigated the anti-lymphoma activity of lymphoma 6C3HED in 1961. Mashburn and Wriston discovered in 1963 that asparaginase isolated from E. coli prevents tumor development during an experiment (Vimal and Kumar 2017a, b). The discovery of this enzyme’s tumor-like property aided in the increased enzyme’s production at industrial scale. It has been used in chemotherapy for over 40 years to treat various cancers, most notably lymphosarcoma lymphoblastic malignancies (Singh and Srivastava 2012). The FDA and the World Health Organization have recommended l-asparaginase therapies for acute lymph leukemia and lymphatic sarcoma (Patil et al. 2012). Additionally, it is used to treat acute myeloblastic leukemia, persistent lymphocytic leukemia, Hodgkin’s disease, melonosarcoma, non-lymphoma, Hodgkin’s pancreatic carcinoma, and bovine lymphosarcoma (Abd El Baky and El Baroty 2016; Warangkar and Khobragade 2009). l-asparaginase has been used as a medication in three distinct ways. Asparaginases (from E. coli), Erwinia asparaginase, and pegaspargase are all members of this community (Priya et al. 2011). In 2016, l-asparaginase recombinant from E. coli was introduced. It may be administered only intravenously, unlike the preceding three can be administered intravenously or intramuscularly (Lanvers-Kaminsky 2017). Commercially active enzymes include Cristanaspase (United States), Crasntin (Germany), Leumase (Japan), Oncaspar, Erwinase, Kidrolase, Crisantas, Pasum, PEG asparaginase, and Pegásparagasum. Both medications are used in the formulation, along with those such as methotrexate, vincristine, and prednisone (Doriya et al. 2016).

Infectious disease

According to a recent report, l-asparaginase prevents the development of Group A streptococcus (GAS) in both human blood and a mouse model of bacterial infection. GAS is a streptococcus-pyogenes bacterium that can induce a variety of infections. Pharyngitis, glomerulonephritis, bone scarlet disorder, traumatic shock syndrome, meningitis, and rheumatic fever are only a few examples. GAS attaches to a host cell and produces streptolysins (streptolysin O and streptolysin S), which induce endoplasmic reticular tension. This enhances asparagin synthesis expression and efficiency, resulting in a high level of l-asparagin in the blood. When l-asparagine levels are increased, GAS gene expression is enhanced, resulting in GAS growth and virulence in the host (Baruch et al. 2014a, b). l-asparaginase release (as a result of GAS infection) can be inhibited to prevent infections in the host. Additionally, this discovery refers to biochemicals that can be targeted for effective pathogen control during pathogenic invasions. According to scientists, other Gram-positive bacteria that produce streptolysin toxins, such as Staphylococcus aureus, Listeria monocytogenes, or Clostridium botulinum, may take the same path. As a result, l-asparaginase can be used to treat an infection that has been caused (Baruch et al. 2014a, b).

Diseases caused by the immune system

In clinical trials, l-asparaginase tends to have immunosuppressive and anti-inflammatory properties. It has a significant inhibitory effect on B-cell responses mediated by T cells. Due to its impact on the lymphatic system, it is most likely to be used to treat inflammatory diseases induced by abnormal T cell responses. Male DBA/1 mice with collagen-induced arthritis (CIA) were treated with E. coli pegylated l-asparaginase. The enzyme was discovered to be highly successful in preventing arthritis in CIA mice. In a comparative study, it was shown to be more efficient and less harmful than the standard drug cyclophosphamide in managing CIA. The thesis examines the ability of this enzyme to be used in the future to cure inflammatory diseases such as rheumatoid arthritis (Reiff et al. 2001; Vimal and Kumar 2018).

Food industry

Apart from its clinical and nutritional benefits, l-asparaginase is employed as a packaging tool in the foodstuff sector. It is used to eliminate acrylamide from fried and baked goods. The International Cancer Research Agency classified acrylamide as “possibly carcinogenic to humans” in multiple studies (IARC paper, 1994). (Vimal and Kumar 2018). Acrylamide is a chemical formed in a variety of starchy foods when cooked at high temperatures above 120 °C, such as when they are fried, grilled, or baked. It is formed when the free amino group of the amino acid asparagine reacts with the carbonyl groups of reducing sugar and is used in a wide variety of starchy foods. This is referred to as the “Millard reaction” (Vimal and Kumar 2017a, b). Before baking or frying, asparaginase is added to prevent the acrylamide from aggregating and converting asparagine to aspartic acid and ammonium ions. As a result, it significantly reduces the amount of acrylamide used as a food-processing aid. It does not affect the finished product’s flavor or quality (Batool et al. 2016). Though, full acrylamide clearance is impractical due to other small asparagine-independent pathways (Zolfaghar et al. 2016). Under the trading names asparaginase, it is available to remove acrylamide from starchy foods. Acrylaway and PreventASe are two such items. The food industry is currently investigating alternative forms of this enzyme to mitigate the effects of acrylamide while retaining its beneficial possessions. The enzyme must be firm over extensive pH and temperature spectrum to reduce processing time while maintaining a high conversion rate and substrate specificity. Additionally, the mechanism has little impact on the other food components (Krishnapura and Belur 2016). Since treatment with acrylate, the percentage of acrylamide in various foods decreased. Another application in the food industry is to impart flavored seasoning to the food through the aspartic acid provided during asparagine hydrolysis. Bacillus subtilis is extensively employed in the food sector to perform this function (Yano et al. 2008).

Chemotherapy for dogs and cats

Lymphoma is the supreme communal form of carcinoma in dogs and cats. Laterally with human lymphomas, l-asparaginase is employed to combat these lymphomas. It is employed with cyclophosphamide and prednisone in multidrug-resistant canine cancer therapy (Saba et al. 2009). In the bulk of cured evidence, canine carcinoma deteriorates, necessitating salvage therapy. Relapses can occur when cytotoxic drug meditations are inadequate at different anatomic localities, such as the CNS. A veterinary oncologist uses l-asparaginase to shrink tumors, allowing physicians to investigate potential therapeutic methods (Zandvliet 2016). However, due to its high expense, it is not often used in veterinary medicine.

Additional applications

It has been discovered that some homo-oligomeric proteins’ monomers exist in a variety of conformations. When these monomers (of the same type) are mixed, a distinct quaternary structure is created. Each quaternary structure of the protein serves a distinct function. Morpheein is the generic term for a variety of proteins. Reversible quaternary rearrangement produces new allosteric locations in these morphines, which may be used as a pharmaceutical target (Selwood and Jaffe 2012). Dimer, tetramer, and inactive octamer types of l-asparaginase occur. As Type-I l-asparaginase binds l-Asparagine, an allosteric reorganization of the tetramer occurs. The complexities of the quaternary arrangement of proteins can be used to discover new medicines (Vimal and Kumar 2017a, b). Numerous conditions have been linked to l-asparaginase as a virulence factor. The usage of Salmonella typhimurium to induce infections and prevent host immune system clearance requires the use of l-asparaginase-II-mediated T cell immunosuppression (Kullas et al. 2012). L-Asparaginasis is a virulence factor generated by Helicobacter pylori, the bacteria that trigger chronic gastritis, peptic ulcers, gastric cancer, and lymphoma of the stomach mucosa. It spreads infection by messing with the host’s cell cycle (Scotti et al. 2012). Mycobacterium tuberculosis utilizes the asparagine transporter AnsP2 and stowed asparaginase AnsA to withstand acidic tension and pH buffering within the macrophages. In each of these instances, it is essential to study the morpheein model of l-asparaginase allosteric regulation to improve the current therapies (Gouzy et al. 2014).

Conclusions

l-asparaginase is a critical chemotherapeutic agent in the medical sector. Numerous experimental studies support the usage of this compound in the management of bacterial and autoimmune diseases. The use of this enzyme as a food-processing aid is well known. Due to the enzyme’s multiple benefits and strong market demand, it must operate at a high-efficiency level while being cost efficient. Immobilized enzymes are increasingly being used in medical, industrial, and nutritional applications. Therefore, research to extend the half-life and stabilize the enzyme is critical, as new carriers and immobilization methods are needed to address this phenomenon. At this stage, knowledge and expertise from the past and present are crucial. Nanoparticles, nanotubes, mesoporous compounds, and nanofibrous membranes have been subjected to nanotechnology studies to immobilize enzymes on their surfaces. Almost all nanoscale carriers for l-ASNase immobilization are briefly discussed in this paper. Future research can lead to the development of improved formulations for l-ASNase.