L-asparaginase production review: bioprocess design and biochemical characteristics

In the past decades, the production of biopharmaceuticals has gained high interest due to its great sensitivity, specificity, and lower risk of negative effects to patients. Biopharmaceuticals are mostly therapeutic recombinant proteins produced through biotechnological processes. In this context, L-asparaginase (L-asparagine amidohydrolase, L-ASNase (E.C. 3.5.1.1)) is a therapeutic enzyme that has been abundantly studied by researchers due to its antineoplastic properties. As a biopharmaceutical, L-ASNase has been used in the treatment of acute lymphoblastic leukemia (ALL), acute myeloblastic leukemia (AML), and other lymphoid malignancies, in combination with other drugs. Besides its application as a biopharmaceutical, this enzyme is widely used in food processing industries as an acrylamide mitigation agent and as a biosensor for the detection of L-asparagine in physiological fluids at nano-levels. The great demand for L-ASNase is supplied by recombinant enzymes from Escherichia coli and Erwinia chrysanthemi. However, production processes are associated to low yields and proteins associated to immunogenicity problems, which leads to the search for a better enzyme source. Considering the L-ASNase pharmacological and food importance, this review provides an overview of the current biotechnological developments in L-ASNase production and biochemical characterization aiming to improve the knowledge about its production. • Microbial enzyme applications as biopharmaceutical and in food industry • Biosynthesis process: from the microorganism to bioreactor technology • Enzyme activity and kinetic properties: crucial for the final application


Introduction
Recent advances in enzyme technology empowered scientists to use, modify, and improve the efficiency of enzymes, leading to their maximum functionality (Muneer et al. 2020). L-asparaginase (L-asparagine amidohydrolase, L-ASNase) (EC 3.5.1.1) has been extensively used and studied because of its relevant potential as an oncological agent and as an acrylamide mitigation agent in the food industry, which is due to its ability to catalyze the hydrolysis of L-asparagine into Laspartate and ammonia (Sharma et al. 2018;Chand et al. 2020). The discovery and development of potential uses of L-ASNase as an anti-cancer drug started in 1953, when Kidd first observed that lymphomas in rat and mice relapsed after treatment with guinea pig serum (Kidd 1953). Nowadays, L-ASNase is widely used in the treatment of acute lymphoblastic leukemia (ALL), acute myeloblastic leukemia (AML), and other lymphoid malignancies in combination with other drugs (Husain et al. 2016;Vala et al. 2018). However, formulation of this protein represents 40% of the total enzyme demands worldwide and one third of the global needs for anticancer agents, which is far more than for other therapeutic enzymes (Izadpanah et al. 2018). The biopharmaceutical world market, especially the healthcare market, is constantly growing. Therapeutical enzyme market was expected to reach $6.3 billion by 2021 as compared to $5.0 billion in 2016, corresponding to a 4.7% annual growth rate for the period (Chand et al. 2020).
L-ASNase is also widely used in food processing industries as an acrylamide mitigation agent since this compound is being classified as a probable carcinogen compound according to several agencies, namely the International Agency for Research on Cancer (Javier et al. 2016). Additionally, L-ASNase biosensor is a promising technology for the detection of L-asparagine in physiological fluids at nano-levels (Batool et al. 2016).
Several microorganisms and even a few plants and animals are endowed with L-ASNase producing ability. However, due to the complex process of extracting and purifying enzymes from plants and animals, the use of microorganisms is the most viable alternative (Moguel 2018). For instance, all L-ASNase drugs commercially authorized for clinical purposes are restricted to recombinant L-ASNase from Escherichia coli and Erwinia chrysanthemi (Muneer et al. 2020). Moreover, in order to have commercial and therapeutic value, L-ASNase must be stable over a wide range of pH and temperature and must have a low Michaelis-Menten constant (K m ) value (high substrate affinity in physiological conditions) and low collateral effects (Chand et al. 2020). Therefore, several investigations have been carried out in order to produce recombinant L-ASNase with improved characteristics and properties. Figure 1 presents an overview of L-ASNase production by recombinant microorganisms.
This review provides a deep overview of the literature regarding the microbial production of L-ASNase using different strategies, as well as its biochemical characterization.

Classification of L-asparaginase
Although L-ASNase can be produced by several sources, this enzyme is classified based on its amino acid sequence, biochemical properties, and structural and functional homology (Müller and Boos 1998). Currently, L-ASNases are divided into three major groups: (i) bacterial type L-ASNase (including the classification type I and type II), (ii) plant type L-ASNase (type III), and (iii) rhizobial type L-ASNase (Borek and Jaskólski 2001;Qeshmi et al. 2018). Bacterial type L-ASNases are structurally and evolutionarily distinct from the plant type (Michalska and Jaskolski 2006). The bacterial type L-ASNases are subdivided in types I and II based on their cellular localization and on the activity towards L-asparagine and L-glutamine (Izadpanah et al. 2018). L-ASNase type I is a cytosolic enzyme with relatively low affinity for L-asparagine and high specific activity towards L-glutamine. On the other hand, type II is a periplasmic or membrane associated L-ASNase with high affinity for L-asparagine and low activity towards L-glutamine, a combination highly attractive for oncologic application (Izadpanah et al. 2018). Both types of L-ASNase can be produced by the same microorganism; for example, E. coli is able to produce two isozymes of L-ASNase (Qeshmi et al. 2018). However, only the L-ASNase type II possesses anti-tumor activity. The characteristics of bacterial L-ASNase type I and type II are summarized in Fig. 2.
The structural feature differentiating type I and II isoenzymes is the active complex size. L-ASNase type I seems to form dimers (Yao et al. 2005;Yano et al. 2008), whereas the L-ASNase type II is widely reported as a tetramer. According to Aghaiypour et al. (2001) and Lubkowski et al. (2003) bacterial L-ASNases type II are active as homo-tetramers with 222 symmetry, each monomer consisting of about 330 amino acid residues forming 14 β-strands and eight α-helices, as shown in Fig. 3 for the three-dimensional structure of Fig. 1 Overview of Lasparaginase production by recombinant microorganisms E. chrysanthemi L-ASNase type II (Lubkowski et al. 2003). Studies on L-ASNase type-I structure are much scarcer in the literature than those for the type II.
It is also important to discuss the structure of L-ASNase at molecular levels. Commonly, the enzyme is found as a tetramer, but monomeric, dimeric, and hexameric forms have also been found for enzymes isolated from different sources (Batool et al. 2016). In fact, molecular structures of L-ASNases from E. coli and Erwinia sp. have been deeply investigated. The native L-ASNase type II isolated from E. coli has a molecular weight of 138-141 kDa and contains four identical subunits of 326 amino acids with one active center each (Kozak and Jurga 2002). The reported molecular weight of the Erwinia-derived L-ASNase is 138 kDa as described in Table 1 (Nguyen et al. 2016;Müller and Boos 1998). Information about the kinetic parameters of this L-ASNase formulation is also presented in Table 1. Different sources and post-translation modifications may strongly influence the molecular structure of the enzyme. For instance, Asselin et al. (1995) reported a PEG-modified L-ASNase from E. coli with increased half-lifetime (5-7 days) and molecular weight of 145 kDa.
The stability and half-lifetime of L-ASNase in the serum are of crucial concern for the pharmaceutical industry. An enzyme preparation with high stability and increased halflifetime can avoid the need for multiple dose administration, which may lead to less chances of triggering hypersensitivity reactions (Krishnapura et al. 2016). Therefore, from the different L-ASNases analyzed for clinical trials, the ones from E. coli modified with PEG revealed a higher half-life when compared to non-modified E. coli L-ASNase, which ensure adequate serum enzyme activity and prevents complete L-ASNase serum depletion (Pieters et al. 2011;Asselin et al. 1995). An often used modification to prevent hypersensitivity Three-dimensional structure of Erwinia chrysanthemi L-asparaginase type II suggested by Aghaiypour et al. (2001) and Lubkowski et al. (2003) reactions towards the native forms of L-ASNase is the PEG conjugation (Müller and Boos 1998;Pui et al. 2018). For preparation of the modified enzyme, units of monomethoxy PEG are attached to the derived enzyme (e.g., E. coli) by covalent bonds (Yoshimoto et al. 1986).

Sources of L-asparaginase
Production of L-asparaginase by wild-type species As cited previously, L-ASNase is widely distributed in nature, being found in animals (fishes, mammals, and birds), in different tissues (such as liver, pancreas, brain, kidneys, and lungs), plants, and microorganisms, including bacteria, filamentous fungi, and yeast Brumano et al. 2019). However, as indicated by Savitri and Wamik, microorganisms are a better source of L-ASNase, considering their ability to grow easily on very simple and economical substrates (Savitri and Azmi 2003). Additionally, the biotechnological production process is usually easier to optimize and scale-up than other processes. Depending on the strain employed, it can be easily genetic modified in order to increase the yield, making the extraction and purification process economically feasible (Cachumba et al. 2016;Lopes et al. 2017). Table 2 shows several microbial wild-type species able to produce L-ASNase and the corresponding enzyme characteristics. The best producers of L-ASNase belong to the Enterobacteriaceae family, followed by fungi species. The main bacteria producers of L-ASNase are E. coli and E. chrysanthemi. However, the main problem associated with L-ASNase produced by prokaryotic microorganism are hypersensitivity and immune inactivation (Javier et al. 2016). In this sense, different strategies with bacterial source have been studied and it will be further discussed in the "Production of recombinant L-asparaginase" section. Considering the production process, the L-ASNase produced by E. coli is intracellular, which inserts the unit operation of disruption cell in the downstream processes. Among the bacteria, wild types of Bacillus are also natural producers of L-ASNase, i.e., Bacillus australimaris NJB19 (MG734654) (Chakravarty et al. 2021), Bacillus lichenformis ( (Mahajan et al. 2014), and Bacillus sp. (Singh and Srivastava 2012).
Other sources of L-ASNase are Actinomycetes strains, which are filamentous bacteria well known as a good source of antibiotics, with microorganisms such as Streptomyces griseoluteus, Nocardia levis, and Streptomyces ginsengisoli reported to be potential producers of L-ASNase (LopesOrabi et al. 2019;Qeshmi et al. 2018). The L-ASNase produced by actinomycetes is generally extracellular, which is an advantage for the production process. Saxena et al. (2015) studied 240 actinomycetes being 165 positives for L-ASNase activity. Among them, the strains Streptomyces cyaneus (SAP 1287, CFS 1560), Streptomyces exfoliates (CFS 1557), and Streptomyces phaeochromogenes (GS 1573) were Lglutaminase-free actinomycetes with a highlighted production of glutaminase-free L-ASNase by the last strain (Saxena et al. 2015). However, studies performed by Dhevagi and Poorani (2006) showed that L-ANSase from marine actinomycetes presented cytotoxic effect on acute T-cell leukemia and myelogenous leukemia, being this source of L-ASNase an alternative for the food industry.
Fungal L-ASNases are commonly produced extracellularly, simplifying the downstream purification process (Chand et al. 2020). Fusarium, Aspergillus, and Penicillium strains are the most common fungi genera reposted to produce L-ASNase (Orabi et al. 2019). L-ASNases from Aspergillus oryzae and Aspergillus niger are already commercially approved for use as processing agents in the food industry (Chand et al. 2020). The health sector requires a nobler source of L-ASNase with minimal or no cross-reactivity to minimize adverse reactions. Alike human cells and unlike bacterial cells, fungi cells can glycosylate proteins; therefore, enzymes isolated from fungi are expected to cause less immunogenicity (Chand et al. 2020). However, the fungal complex morphology can be critical for the feasibility of scaling up the process since fungal cultivation in bioreactor is sensible to several parameters, such as oxygen supply and transfer, inoculum size, pH, and stirring (De Oliveira et al. 2020).
Several yeast genera (Table 2), including Saccharomyces, Candida, Pichia, Rhodotorula, Rhodosporidium, and Trichoderma, have been reported as L-ASNase producers (Chand et al. 2020;Kil et al. 1995). Saccharomyces cerevisiae strains were found to produce both the intracellular and extracellular forms of L-ASNase, whereas production of the extracellular form seems to be triggered under nitrogen starvation (Sharma et al. 2018). An alternative source of L-ASNase is blue-green microalgae, an attractive option due to its no seasonal variation, low cost of medium formulation, and easy cultivation and harvesting characteristics (Orabi et al. 2019). Chlamydomonas sp., Chlorella vulgaris, and Spirulina maxima are considered as potential microalgal sources for novel enzyme production in several studies (Orabi et al. 2019;Ebrahiminezhad et al. 2014;Abd El Baky and El Baroty 2016).
As demonstrated, several microorganisms presenting particular characteristics can act as potential producers of L-ASNase. However, before designing and scaling up the bioprocess, pharmaceutical and food industries seek for high productivity, easily handling and scaling up, highly stable enzymes (temperature, pH, storage), high enzymatic activity, low toxicity, easy product purification, and low production costs (Chand et al. 2020;Brumano et al. 2019). Therefore, in-depth studies are essential to disclose the best sources of the enzyme for industrial applications.

Production of recombinant L-asparaginase
Recently, many efforts to produce recombinant L-ASNase from different sources have been made, as depicted in Table 3. Each system presents its own characteristics regarding production capacity, cost, safety, complexity, and processing impact . It is important to highlight that in the last years there was significant progress in synthetic biology through the development of molecular tools and methods for engineering biological systems, which facilitate the construction of efficient chassis for industrial relevant bioprocesses, including the production of L-ASNase (Corrêa et al. 2020). To exemplify, Corrêa et al. (2020) presented the engineering of tunable and modular devices for autonomous control of gene expression in Bacillus subtilis that requires no inducer and no human supervision. The device developed can be applied for heterologous protein production (Corrêa et al. 2020).
The preferred host for overproduction of recombinant L-ASNase is E. coli, and the pET system with isopropyl β-Dthiogalactoside (IPTG) induction is the most used gene expression system (as summarized in Table 3). However, additional work has been done on alternative hosts such as B. subtilis (Feng et al. 2017;Sushma et al. 2017;Li et al. 2018;Niu et al. 2021) and Pichia pastoris (Sajitha et al. 2015;Rodrigues et al. 2019;Lima et al. 2020). Unlike E. coli, these hosts hold the GRAS (Generally Regarded as *nd not determined. **International unit (IU) of asparaginase activity is defined as the amount of enzyme required to release 1 μmol of ammonia per minute at specified conditions Safe) status, and can be engineered to secrete the enzyme to the medium, which may turn the downstream process easier and of lower cost. From the gene sequence it is possible to establish that the ansZ gene from B. subtilis encodes a L-ASNase with 59% identity to the L-ASNase type I from E. chrysanthemi and 53% identity to the L-ASNase type II from E. coli (Fisher and Wray 2002). Moreover, B. subtilis has another gene (ansA) that encodes a L-ASNase type I (Yano et al. 2008). Feng et al. (2017) were able to successfully overproduce and secrete a recombinant L-ASNase type II in B. subtilis, reaching 2.5 g L −1 of enzyme in a 3-L bioreactor through a fed-batch strategy. More than protein secretion, P. pastoris is able to add post-translation modifications to the overproduced enzyme. Lima et al. (2020) used P. pastoris to engineer a L-ASNase with a human-like glycosylation pattern, which lowered the immunogenicity of the protein tested in vitro compared to the non-glycosylated.

Biochemical characterization of L-asparaginase
Effect of pH and temperature in L-asparaginase activity In order to guarantee the best possible performance of an enzyme, biochemical characterization regarding temperature and pH are essential parameters to define its application (Krishnapura et al. 2016). Different studies have been performed in order to evaluate the effect of pH on activity of L-ASNase produced by different microorganisms (Table 4). In general, the L-ASNase maximum activity ranges from acidic to alkaline pH values (Chand et al. 2020). The pH affects not only the enzyme structure but also its affinity for the substrate. For therapeutic use, optimal pH for the L-ASNase must lie in the physiological range, while for the food industry, the L-ASNase must keep enough activity even at acidic pH (Krishnapura et al. 2016). L-ASNase produced by bacteria such as E. coli, Bacillus megaterium, and Pseudomonas fluorescens presents optimum activity at pH of 6.0, 7.0, and 7.5, respectively (Borah et al. 2012;Zhang et al. 2015;Sindhu and Manonmani 2018b). According to Jeyaraj et al. (2020), a pH value close to 8.0 is needed for a maximum activity for L-ASNase from B. subtillis (Jeyaraj et al. 2020). The enzyme produced by Penicillum sp. and Anoxybacillus flavithermus, a fungus and a bacterium, respectively, both demonstrate an optimal activity at pH of 7.0 (Chand et al. 2020;Maqsood et al. 2020). On the other hand, the Gram-negative bacteria Pseudomonas aeruginosa PAO1 and Rhizobium etli produce enzymes with maximum activity in acidic and alkaline conditions, 5.5 and 9.0, respectively (Angélica et al. 2012;Dutta et al. 2015).
Temperature also affects the pace of catalysis and stability of an enzyme (Daniel et al. 2010). Temperature tolerance and stability of L-ASNases differs from species to species (Table 4); however, the enzymes often have optimal activity in a temperature range between 25°C and 45°C (Chand et al. 2020). Nevertheless, the extreme thermophiles Thermococcus kodaka (TK1656) and T. kodaka (TK2246) produce L-ASNases with optimal activity at 85°C (Chohan et al. 2020;Muneer et al. 2020). In the study performed by Kumar et al. (2017), the authors concluded that the L-ASNase produced by B. subtillis shows an optimal activity at 37°C. Additionally, authors showed that this enzyme is also active in a wide range of temperature from 30°C to 75°C; yet, at the maximum temperature will eventually lead to an unstable enzyme with no application . Similarly, Patro and Gupta (2012) obtained a L-ASNase from Penicillum sp. with optimal activity at 37°C. The authors determined the optimal temperature for the enzyme using a range of temperatures between 30°C and 50°C. The study performed by Borah et al. (2012) shows the production of L-ASNase from E. coli, whereas the optimal enzyme activity was achieved at 55°C. As well in this study, the authors defined that the enzyme produced was able to tolerate high temperatures and hence can be considered a thermostable enzyme (Borah et al. 2012).
One way to preserve and/or improve the enzyme characteristics including L-ASNase activity and stability is to confine or to immobilize the enzyme in nanomaterials. The process can enhance thermal, pH, storage, and operational stabilities, and can even improve the pharmacological properties, as high enzyme selectivity. This modification process may also prevent enzyme deactivation . Cristovão et al. (2020) studied the application of multi-walled carbon nanotubes (MWCNTs) as support for ASNase immobilization by adsorption method. According to the results, MWCNTs are efficient supports for ASNase immobilization, with no chemical modification or covalent binding required, opening up the possibility for ASNase-MWCNT bioconjugates in several applications. L-ASNase immobilization and confinement techniques are interesting to maintain the enzyme biochemical properties.

Influence of effector molecules on L-asparaginase activity
Metal ions are essential for the structural regulation of a protein as they act as electron donors or acceptors (Buchholz et al. 2012). In some cases, the presence of a metal ion is mandatory for the preservation of the multimeric structure of the enzyme and also to stabilize the reaction intermediates (Krishnapura et al. 2016). For a better understanding of the mechanism of enzyme action it is important that the influence of various effectors that activate or inhibit (or in any other way affect) the protein is well described and studied. These data may lead  The same metal chelator can have different influence on L-ASNase isolated from different sources. Ethylenediamine tetra acetic acid (EDTA) enhances the activity of the enzyme from Erwinia carotovora but has no effect on the L-ASNase from Cladosporium sp. (Krishnapura et al. 2016). Divalent ions, such as Ca 2+ , Co 2+ ,Cu 2+ ,Mn 2+ , Hg 2+ , Mg 2+ , Fe 2+ , Sn 2+ , Pb 2+ , and Ba 2+ , were proved to have an inhibitory effect on L-ASNase from Bacillus aryabhattai ITHBHU02, while Na + and K + enhance the enzymatic activity (Singh et al. 2013). For L-ASNase from Thermococcus gammatolerans EJ3, Mg 2+ acts as an activator, while Zn 2+ , Co 2+ , Ca 2+ , Mn 2+ , Ni 2+ , Cu 2+ , and Ba 2+ are considered inhibitors of the enzyme (Zuo et al. 2014).

Kinetic properties of L-asparaginase
Efforts to produce recombinant L-ASNases and the search for new different wild sources are mostly directed towards developing alternatives for treating ALL patients that develop hypersensibility reactions to the available commercial L-ASNase. The underlining idea is that enzymes from different sources provide different protein sequences that may present different immunogenicity profiles. However, there are other requirements for a new enzyme to become an efficient new oncogenic biopharmaceutical, such as the kinetic parameters. Because L-asparagine is present at~50 μM in the human blood, therapeutic L-ASNase must have a substrate affinity in the lower micromolar range (Ollenschläger et al. 1988;Nguyen et al. 2016). Low Michaelis-Menten constant (K m ) associated with high turnover number (K cat ) ensure that the therapeutic L-ASNase will sufficiently reduce the endogenous L-asparagine at safe doses (Beckett and Gervais 2019). Apart from this, kinetic parameters are crucial for the efficient use of enzymes in different industrial processes (Choi et al. 2017). Most of the mesophilic L-ASNase reported to date have low K m values while the thermophilic ones show relatively high K m (Hong et al. 2014). The values of kinetic parameters for L-ASNases obtained from wild type and recombinant microorganisms are listed in Table 2 and Table 3, respectively.
In that regard, few promising sources of recombinant enzymes were recently characterized: L-ASNase type II from     (Krishnapura et al. 2016).
Regarding wild-type microorganism, several authors have reported L-ASNases with elevated substrate affinity; for example, Warangkar and Khobragade (2010) produced an efficient enzyme from Erwinia carotovora presenting K m value of 0.096 mM. Elevated substrate affinity was also obtained by Mahajan et al. (2014) when studying the enzyme produced by Bacillus licheniformis, presenting a K m = 0.014 mM (Mahajan et al. 2014). For instance, Asha and Pallavi (2012) reported an enzyme from Fusarium sp. presenting V max = 40 IU and K m = 443.98 mM and indicated its potential in cancer therapy since the enzyme did not elicit any immunostimulatory response in human lymphocytes in vitro, unlike most of the reported prokaryotic asparaginases (Asha and Pallavi 2012). Enzymes generally present complex action mechanism systems and need to be deeply studied before efficient and safe application. Kinetic characterization has a key role in understanding enzyme activity and in designing the most efficient application routes. Additionally, as shown in Tables 2 and 3, the values of K m and K cat are intrinsically related to the enzyme source and represent important comparison parameters in order to evaluate the potential application of the protein.

Bioprocess for L-asparaginase production
The effective application of a bioprocess for the production of the target enzyme requires a meticulously selection of the microorganism as the basis of the process, as it affects directly the characteristics of the final product (Brumano et al. 2019). Among the different species capable of producing L-ASNase and as previously mentioned, E. coli is the main microbial host used for the industrial-scale production of recombinant L-ASNase. However, other species have been studied and are promising candidates. L-ASNase production can be performed by submerged fermentation (SmF) and solid state fermentation (SSF) . Figure 4 summarizes the main advantages and limitations for both fermentation process types.
SmF is the main type of fermentation employed for bacterial enzyme production and, consequently, the most used to produce L-ASNase. In fact, SmF is well established and the manipulation of medium components is comparatively easier, leading to high production yields (Vimal and Kumar 2017). Moreover, no requirement for pre-treatment of substrate, easiness of manipulation of the reaction parameters and easy purification of products strongly contribute for the widely application of this type of fermentation. This type of fermentation allows the microorganism to grow in closed reactors containing a liquid broth medium. High concentration of dissolved oxygen is usually required (Doriya et al. 2016).
As for other biomolecules, the process to obtain L-ASNase is considerably influenced by several factors, such as type and concentration of carbon and nitrogen sources, pH, temperature, fermentation time, aeration, and mainly the microbial agent . The productivity of microbial metabolites is related to the process variables such as type and concentrations of nutrients, and operation conditions (Marques et al. 2014). Submerged fermentations can be performed in laboratory scale (shaken flasks culture and bioreactor up to 10 L) and industrial scale (bioreactor larger than 10 L). The shaken flask experiments are important to study the performance of microorganisms with minimal costs and material; therefore, it is extensively used to optimize some conditions for the biotechnological process, such as carbon and nitrogen source and concentration, microelement presence, among others. However, the production in shaker incubator presents several limitations such as limited oxygen transfer, and inability to control pH and dissolved oxygen tension. Moreover, for industrial application, high amount of product is necessary and the production in bioreactor can improve the process reducing the product final cost (Gamboa-Suasnavart et al. 2013).
In bioreactor, the operation mode can generate high productivities. It can be carried out as batch (all nutrients required for the culture are added at the beginning of the cultivation, whereas the product, by-products, and non-consumed components are removed at the end of each batch), fed-batch (some nutrients are provided during the process until a limitation of volume, and the product is removed at the end of each batch), and continuous fermentation (nutrients are added continuously, and product is removed at the same speed of the feeding flow, with the volume inside the bioreactor remaining constant) (Torres et al. 2016). Currently, there are several reports exploring the production of L-ASNase in shaken flasks and a few in bioreactor. However, with the market need for this enzyme, further studies in bioreactor are mandatory. To exemplify how the production step is important, de Oliveira et al. (2019) studied the production of natural colorants with antimicrobial properties, obtaining a 30-fold increase varying only the culture media in shaken flask experiments after 168 h of bioprocess. Later on, the same authors working with bioreactor stirred tank under batch cultivation reduced the time of bioprocess for 120 h, achieving similar amount of colorants (De Oliveira et al. 2020). Regarding L-ASNase production, Kumar et al. (2011a, b) with Pectobacterium carotovorum MTCC 1428 produced an enzyme with 17.81 IU·mL −1 of activity in shake flask level. Kumar et al. (2011a, b) working with the same microorganism but in batch and fed-batch mode feeding L-asparagine and/or glucose, produced 18 and 38.8 IU·mL −1 , respectively, demonstrating the importance of studies in bioreactor.
Production of L-ASNase from various microbial sources by SmF and the respective optimized conditions reported in the literature are summarized in Table 5.
The most frequently reported culture media for L-ASNase production by SmF are Luria-Bertani (LB) medium, tryptone glucose yeast extract broth, and modified Czapek-Dox medium with optimal pH ranging from 6.2 to 7.5, temperature from 28 to 37°C, and fermentation times ranging from 24 to 168 h depending upon the type of the employed microorganism (Dharmaraj 2011;Usha et al. 2011;Gurunathan and Renganathan 2012;Einsfeldt et al. 2016;Vimal and Kumar 2017;El-Naggar et al. 2018). Using LB broth as medium, a L-ASNase with an activity of 8.7 IU mg −1 and 23.85 IU mg −1 was obtained from Yersinia pseudotuberculosis YpA and B. subtilis hswx88, respectively (Pokrovskaya et al. 2012;Jia et al. 2013).
According to Singh et al. (2013), the production of L-ASNase from B. aryabhattai ITBHU02, using M9 medium and L-asparagine as nitrogen source, reached an enzymatic activity of 9.88 IU mg −1 with optimal temperature and pH of 40°C and 8.5, respectively (Singh et al. 2013). The M9 medium and L-asparagine as nitrogen source was also used by Chakravarty et al. (2021) in the study of L-ASNase production by B. australimaris. The authors performed the experiments in incubator shaker and using Box-Behnken design achieved an enzyme production of 37.93 IU mL −1 at the following conditions: 48 h of incubation time, 35°C, 1.25% (w/v) of inoculum, and 2.5% (w/v) of L-asparagine (Chakravarty et al. 2021). The authors also identified the L-ASNase gene and cloned it in E. coli using pET30b vector and demonstrated that the L-ASNase produced was type II (Chakravarty et al. 2021). Erva et al. (2017) produced L-ASNase from Enterobacter aerogenes MTCC111 with an activity of 18.35 IU mL −1 applying trisodium citrate (0.75% (m/v)) and ammonium chloride (0.15% (m/v)) for 40 h at 33°C (Erva et al. 2017). Using Emericella nidulans to produce L-ASNase, Jayaramu et al.
(2010) obtained a protein with an activity of 1.1 IU mL −1 with fermentation period of 48 h, at 30°C, and pH of 6.0 (Jayaramu et al. 2010). These results indicate that each potential producing strain requires its own specific conditions, and there are no established fixed parameters for Smf. Thus, specific optimization studies need to be performed after the microorganism selection.
As aforementioned, L-ASNase production by SmF from recombinant microbial strains, such as E. coli, has been employed aiming to meet the current market demand. However, as depicted in Table 5, there are several studies performed in SmF with other potential microorganisms that can result in high yields of L-ASNase and these enzymes can be applied in the food industry.
Considering SFF, it emerged as an alternative to SmF for the production of extracellular enzymes as it allows the direct use of crude fermented product as enzyme source and has the potential for the production of secondary metabolites . Generally, this process uses cheap agriculture waste such as rice bran, wheat bran, sesame oil cake, corn cob, soybean meal, gram husk, coconut oil cake, groundnut cake, and tea waste (Vimal and Kumar 2017). The use of agricultural wastes not only makes the procedure less cost effective but also reduces the environmental pollution (Vimal and Kumar 2017). In this fermentation process, substrates are used slowly and steadily by the microorganism. That means the same substrate can be used for long fermentation periods (Nadu 2012). In fact, SSF is more relevant for fermentation processes involving fungi and microorganisms that require less moisture content. It does not suit fermentation processes involving organisms that require a high water activity, such as bacteria (Babu and Satyanarayana 1996;Nadu 2012). Additionally, this process offers benefits such as low energy and equipment requirement, cheaper growth substrates, and the downstream processes can be easier since the fermentation process can provide more concentrated solutions, turning unnecessary the use of concentrating unit operations (Holker and Lenz 2005). However, when compared

SUBMERGED FERMENTATION
Superior heat and mass transference.
Industrially accessible in huge scope.
Low profit.
High cost.
Elevated amount of effluents.

Culture media Submerged FermentaƟon
Air Microorganism L-ASNase

SOLID-STATE FERMENTATION
Resistance to contaminaƟon.
Challenging process parameters.
Heat build up. Dharmaraj (2011) with SmF, only few reports are available on SFF (Table 6) for the L-ASNase production. Venil and Lakshmanaperumalsamy (2009) produced an L-ASNase with an activity of 79.84 IU g −1 using a modified strain of Serratia marcescens grown in rice bran for 36 h with 50% moisture, at 30°C, and pH of 7.0 (Venil and Lakshmanaperumalsamy 2009). Suresh and Raju (2012) optimized the production of L-ASNase by SSF from Aspergillus terreus MTCC 1782 using different culture media, such as sesame oil cake (SOC), black gram husk (BH), and a mix of both, with temperature and moisture ranging from 30°C to 32°C , and 40% to 60%, respectively. The fermentation period ranged between 96 h and 120h. The optimal culture medium ended up being the mixture of SOC and BH (7:3), reaching an enzymatic activity of 163.34 IU g −1 , while using just black gram husk resulted in an enzyme with 15.95 IU g −1 of activity (Suresh and Raju 2012). Reports from Mishra (2006) revealed an L-ASNase with an activity of 40.9 U g −1 using bran of Glycine max with a 70% moisture for 96 h at 30°C and pH of 6.5 (Mishra 2006

Operating conditions influencing L-asparaginase production
The optimum production period for L-ASNase from microbial sources varies from 24 to 72 h, depending on the microorganism employed. The lowest optimum cultivation time for production of this enzyme was reported in Staphylococcus aureus strain NCTC413 corresponding to a total of 10 h (Chand et al. 2020). On the other hand, among the analyzed studies, the highest optimum period for L-ASNase production bioprocess was registered for Spirulina maxima with 432 h (18 days), which allowed to obtain an activity of 51.28 IU L −1 (Abd El Baky and El Baroty 2016). Among the reported actinomycetes, the maximum enzyme production of 8.79 U mg −1 was obtained after a culture period of 144 h for Streptomyces brollosae NEAE-115 using dextrose starch as production medium (El-Naggar et al. 2018).
Besides the fermentation period, one of the most essential parameters in bioprocessing is the temperature. Optimum temperatures reported for L-ASNase production by most microorganisms ranged from 25°C to 37°C. In fact, cultivation temperature has a direct effect on the development of microorganisms and, consequently, affects the enzyme production and its activity (Ghosh et al. 2013). L-ASNase produced from  Ghosh et al. (2013) demonstrated that Serratia marcescens NCIM 2919 incubated with Citrus limetta pulp showed an optimal temperature of 28°C, while when incubated with a medium made up of coconut oil cake and sesame oil cake revealed an optimal temperature of 35°C and 37°C, respectively, indicating once again the complexity of establishing an optimized industrial bioprocess (Ghosh et al. 2013). Nevertheless, there are organisms like Streptomyces gulbargensis and Fusarium equiseti with higher optimal temperatures (40°C and 45°C, respectively), which when applied to other enzymes results in lower production or lower activity (Amena et al. 2010;Hosamani and Kaliwal 2011). These microorganisms (S. gulbargensis and F. equiseti) can be considered as sources to be explored further for production of heatresistant L-ASNase for food processing. The pH of the culture also affects the bioprocessing of the enzyme alongside the transport of several components across the cell membrane (Chand et al. 2020). The regulation of pH is indispensable while using carbon sources like glucose, fructose, or mannitol once it decreases the pH of the medium. This drop occurs as a result of acid production in the fermentation process which leads to inhibition of L-ASNase production (Alrumman et al. 2019). Several studies report that the optimum pH to produce L-ASNase from bacterial sources is usually close to 7.
According to Moorthy and Sumantha (2010) and Narta et al. (2011), both Bacillus sp. and Bacillus brevis produced L-ASNases with maximal enzyme activity at pH 7.0, revealing a specific activity of 1 IU mg −1 and 2.036 IU mg −1 , respectively (Moorthy and Sumantha 2010;Narta et al. 2011). Prema et al. (2013) demonstrated that Pseudomonas fluorescens produced L-ASNase with an activity of 168.4 IU mL −1 at an optimal pH of 8 (Prema et al. 2013). Regarding to Vibrio species, there are some reports from 70s and 80s such as the bacteria Vibrio succinogenes studied by Kafkewitz and Goodman (1974) which revealed an optimal pH between 7.0 and 7.2 for L-ASNase production using succinate as medium, and Krautheim et al. (1982) which demonstrated that using sodium fumarate and cysteine as medium the optimal pH is 7.2-7.3. Recently, L-ASNase from Vibrio species has been overexpressed recombinantly in Escherichia coli (Radha et al. 2018;Radha and Gummadi 2020).
Overall, new species or recombinant microorganism that overexpress L-ASNase with improved characteristics such as glutaminase free, stability, and promote fewer collateral effects are key factors to enlarge the application of L-ASNase.