A Structural In Silico Analysis of the Immunogenicity of L-Asparaginase from Penicillium cerradense.

L-asparaginase is an essential drug used to treat acute lymphoid leukemia (ALL), a cancer of high prevalence in children. Several adverse reactions associated with L-asparaginase have been observed, mainly caused by immunogenicity and allergenicity. Some strategies have been adopted, such as searching for new microorganisms that produce the enzyme and applying protein engineering. Therefore, this work aimed to elucidate the molecular structure and predict the immunogenic profile of L-asparaginase from Penicillium cerradense, recently revealed as a new fungus of the genus Penicillium and producer of the enzyme, as a motivation to search for alternatives to bacterial L-asparaginase. In the evolutionary relationship, L-asparaginase from P. cerradense closely matches Aspergillus species. Using in silico tools, we characterized the enzyme as a protein fragment of 378 amino acids (39 kDa), including a signal peptide containing 17 amino acids, and the isoelectric point at 5.13. The oligomeric state was predicted to be a homotetramer. Also, this L-asparaginase presented a similar immunogenicity response (T- and B-cell epitopes) compared to Escherichia coli and Dickeya chrysanthemi enzymes. These results suggest a potentially useful L-asparaginase, with insights that can drive strategies to improve enzyme production.

Microbial products for space nutrition.

Sustainably producing nutrients beyond Earth is one of the biggest technical challenges for future extended human space missions. Microorganisms such as microalgae and cyanobacteria can provide astronauts with nutrients, pharmaceuticals, pure oxygen, and bio-based polymers, making them an interesting resource for constructing a circular bioregenerative life support system in space.

PEGylation versus glycosylation: effect on the thermodynamics and thermostability of crisantaspase.

Thermostability is an important and desired feature of therapeutic proteins and is critical for the success or failure of protein drugs development. It can be increased by PEGylation-binding of poly(ethylene glycol) moieties-or glycosylation-post-translational modification to add glycans. Here, the thermostability and thermodynamic parameters of native, PEGylated, and glycosylated versions of the antileukemic enzyme crisantaspase were investigated. First-order kinetics was found to describe the irreversible deactivation process. Activation energy of the enzyme-catalyzed reaction (E*) was estimated for native, PEGylated, and glycosylated enzyme (10.2, 14.8, and 18.8 kJ mol-1 respectively). Half-life decreased progressively with increasing temperature, and longer half-life was observed for PEG-crisantaspase (87.74 min) at 50 °C compared to the native form (9.79 min). The activation energy of denaturation of PEG-crisantaspase (307.1 kJ mol-1) was higher than for crisantaspase (218.1 kJ mol-1) and Glyco-crisantaspase (120.0 kJ mol-1), which means that more energy is required to overcome the energy barrier of the unfolding process. According to our results, PEG-crisantaspase is more thermostable than its native form, while Glyco-crisantaspase is more thermosensitive.

l-Asparaginase Type II from Fusarium proliferatum: Heterologous Expression and In Silico Analysis.

The search for new drug-producing microorganisms is one of the most promising situations in current world scientific scenarios. The use of molecular biology as well as the cloning of protein and compound genes is already well established as the gold standard method of increasing productivity. Aiming at this increase in productivity, this work aims at the cloning, purification and in silico analysis of l-asparaginase from Fusarium proliferatum in Komagataella phaffii (Pichia pastoris) protein expression systems. The l-asparaginase gene (NCBI OQ439985) has been cloned into Pichia pastoris strains. Enzyme production was analyzed via the quantification of aspartic B-hydroxamate, followed by purification on a DEAE FF ion exchange column. The in silico analysis was proposed based on the combined use of various technological tools. The enzymatic activity found intracellularly was 2.84 IU/g. A purification factor of 1.18 was observed. The in silico analysis revealed the position of five important amino acid residues for enzymatic activity, and likewise, it was possible to predict a monomeric structure with a C-score of 1.59. The production of the enzyme l-asparaginase from F. proliferatum in P. pastoris was demonstrated in this work, being of great importance for the analysis of new methodologies in search of the production of important drugs in therapy.

Enhanced production of N-acetyl-glucosaminidase by marine Aeromonas caviae CHZ306 in bioreactor.

N-Acetyl-glucosaminidases (GlcNAcases) are exoenzymes found in a wide range of living organisms, which have gained great attention in the treatment of disorders related to diabetes, Alzheimer's, Tay-Sachs', and Sandhoff's diseases; the control of phytopathogens; and the synthesis of bioactive GlcNAc-containing products. Aiming at future industrial applications, in this study, GlcNAcase production by marine Aeromonas caviae CHZ306 was enhanced first in shake flasks in terms of medium composition and then in bench-scale stirred-tank bioreactor in terms of physicochemical conditions. Stoichiometric balance between the bioavailability of carbon and nitrogen in the formulated culture medium, as well as the use of additional carbon and nitrogen sources, played a central role in improving the bioprocess, considerably increasing the enzyme productivity. The optimal cultivation medium was composed of colloidal α-chitin, corn steep liquor, peptone A, and mineral salts, in a 5.2 C:N ratio. Optimization of pH, temperature, colloidal α-chitin concentration, and kLa conditions further increased GlcNAcase productivity. Under optimized conditions in bioreactor (i.e., 34 °C, pH 8 and kLa 55.2 h-1), GlcNAcase activity achieved 173.4 U.L-1 after 12 h of cultivation, and productivity no less than 14.45 U.L-1.h-1 corresponding to a 370-fold enhancement compared to basal conditions.

Current state of molecular and metabolic strategies for the improvement of L-asparaginase expression in heterologous systems.

Heterologous expression of L-asparaginase (L-ASNase) has become an important area of research due to its clinical and food industry applications. This review provides a comprehensive overview of the molecular and metabolic strategies that can be used to optimize the expression of L-ASNase in heterologous systems. This article describes various approaches that have been employed to increase enzyme production, including the use of molecular tools, strain engineering, and in silico optimization. The review article highlights the critical role that rational design plays in achieving successful heterologous expression and underscores the challenges of large-scale production of L-ASNase, such as inadequate protein folding and the metabolic burden on host cells. Improved gene expression is shown to be achievable through the optimization of codon usage, synthetic promoters, transcription and translation regulation, and host strain improvement, among others. Additionally, this review provides a deep understanding of the enzymatic properties of L-ASNase and how this knowledge has been employed to enhance its properties and production. Finally, future trends in L-ASNase production, including the integration of CRISPR and machine learning tools are discussed. This work serves as a valuable resource for researchers looking to design effective heterologous expression systems for L-ASNase production as well as for enzymes production in general.

Cathepsin B Is Not an Intrinsic Factor Related to Asparaginase Resistance of the Acute Lymphoblastic Leukemia REH Cell Line.

L-Asparaginase (ASNase) is a biopharmaceutical used as an essential drug in the treatment of acute lymphoblastic leukemia (ALL). Yet, some cases of ALL are naturally resistant to ASNase treatment, which results in poor prognosis. The REH ALL cell line, used as a model for studying the most common subtype of ALL, is considered resistant to treatment with ASNase. Cathepsin B (CTSB) is one of the proteases involved in the regulation of in vivo ASNase serum half-life and it has also been associated with the progression and resistance to treatment of several solid tumors. Previous works have shown that, in vitro, ASNase is degraded when incubated with REH cell lysate, which is prevented by a specific CTSB inhibitor, suggesting a function of this protease in the ASNase resistance of REH cells. In this work, we utilized a combination of CRISPR/Cas9 gene targeting and enzymatic measurements to investigate the relevance of CTSB on ASNase treatment resistance in the ALL model cell line. We found that deletion of CTSB in REH ALL cells did not confer ASNase treatment sensitivity, thus suggesting that intrinsic expression of CTSB is not a mechanism that drives the resistant nature of these ALL cells to enzymes used as the first-line treatment against leukemia.

Microbial astaxanthin: from bioprocessing to the market recognition.

The attractive biological properties and health benefits of natural astaxanthin (AXT), including its antioxidant and anti-carcinogenic properties, have garnered significant attention from academia and industry seeking natural alternatives to synthetic products. AXT, a red ketocarotenoid, is mainly produced by yeast, microalgae, wild or genetically engineered bacteria. Unfortunately, the large fraction of AXT available in the global market is still obtained using non-environmentally friendly petrochemical-based products. Due to the consumers concerns about synthetic AXT, the market of microbial-AXT is expected to grow exponentially in succeeding years. This review provides a detailed discussion of AXT's bioprocessing technologies and applications as a natural alternative to synthetic counterparts. Additionally, we present, for the first time, a very comprehensive segmentation of the global AXT market and suggest research directions to improve microbial production using sustainable and environmentally friendly practices. KEY POINTS: • Unlock the power of microorganisms for high value AXT production. • Discover the secrets to cost-effective microbial AXT processing. • Uncover the future opportunities in the AXT market.

L-Asparaginase as the gold standard in the treatment of acute lymphoblastic leukemia: a comprehensive review.

L-Asparaginase is an antileukemic drug long approved for clinical use to treat childhood acute lymphoblastic leukemia, the most common cancer in this population worldwide. However, the efficacy and its use as a drug have been subject to debate due to the variety of adverse effects that patients treated with it present, as well as the prompt elimination in plasma, the need for multiple administrations, and high rates of allergic reactions. For this reason, the search for new, less immunogenic variants has long been the subject of study. This review presents the main aspects of the L-asparaginase enzyme from a structural, pharmacological, and clinical point of view, from the perspective of its use in chemotherapy protocols in conjunction with other drugs in the different treatment phases.

In Silico Design of a Chimeric Humanized L-asparaginase.

Acute lymphoblastic leukemia (ALL) is the most common cancer among children worldwide, characterized by an overproduction of undifferentiated lymphoblasts in the bone marrow. The treatment of choice for this disease is the enzyme L-asparaginase (ASNase) from bacterial sources. ASNase hydrolyzes circulating L-asparagine in plasma, leading to starvation of leukemic cells. The ASNase formulations of E. coli and E. chrysanthemi present notorious adverse effects, especially the immunogenicity they generate, which undermine both their effectiveness as drugs and patient safety. In this study, we developed a humanized chimeric enzyme from E. coli L-asparaginase which would reduce the immunological problems associated with current L-asparaginase therapy. For these, the immunogenic epitopes of E. coli L-asparaginase (PDB: 3ECA) were determined and replaced with those of the less immunogenic Homo sapiens asparaginase (PDB:4O0H). The structures were modeled using the Pymol software and the chimeric enzyme was modeled using the SWISS-MODEL service. A humanized chimeric enzyme with four subunits similar to the template structure was obtained, and the presence of asparaginase enzymatic activity was predicted by protein-ligand docking.

Endochitinase and Chitobiosidase Production by Marine Aeromonas caviae CHZ306: Establishment of Nitrogen Supplementation.

Aeromonas caviae CHZ306, a marine-derived bacterium isolated from zooplankton, can use chitin (a polymer of a ß-(1,4)-linked N-acetyl-D-glucosamine) as a carbon source. The chitin is hydrolyzed by chitinolytic enzymes, namely endochitinases and exochitinases (chitobiosidase and N-acetyl-glucosaminidase). Indeed, the chitinolytic pathway is initiated by the coexpression of the enzymes endochitinase (EnCh) and chitobiosidase (ChB); however, few studies, including biotechnological production of these enzymes, have been reported, although chitosaccharide are helpful in several industries, such as cosmetics. This study demonstrates the potential to maximize the simultaneous EnCh and ChB production by nitrogen supplementation on culture media. Twelve different nitrogen supplementation sources (inorganic and organic) previously analyzed in elemental composition (carbon and nitrogen) were tested and evaluated in the Erlenmeyer flask culture of A. caviae CHZ306 for EnCh and ChB expression. None of the nutrients inhibited bacterial growth, and the maximum activity in both EnCh and ChB was observed at 12 h, using corn-steep solids and peptone A. Corn-steep solids and peptone A were then combined at three ratios (1:1, 1:2, and 2:1) to maximize the production. The high activities for EnCh (30.1 U.L-1) and ChB (21.3 U.L-1) were obtained with 2:1 corn-steep solids and peptone A, corresponding to more than 5- and 3-fold enhancement, respectively, compared to the control condition.

A scoping review to compare and contrast quality assurance aspects of l-asparaginase biosimilars.

l-asparaginase is a first-line medicine used for the treatment of acute lymphoblastic leukemia. Differing quality of marketed l-asparaginase biosimilars has been reported to adversely influence treatment outcomes. Herein, the quality of l-asparaginase biosimilars intended for clinical use was reviewed in sight of quality assurance parameters using English and Chinese language database searching, which provided information for possible improvements to the manufacture of this medicine. Ten articles met inclusion criteria, and quality attributes that measured potency, specific activity, purity and host cell proteins (HCPs) were identified. Biosimilars manufactured in high-income countries represented good quality in all aspects. Biosimilars manufactured in high-middle/middle-income countries, however, suggested poorer quality control particularly over removal of HCPs. Future work should now focus on establishing pharmacopeia monographs to establish equivalent quality assurance for l-asparaginase biosimilars manufactured between countries. Standardization of the quality profile, analytical methods and the limits of critical quality parameters, are essential to ensure appropriated efficacy and safety of clinical grade l-asparaginase.

Insights into using green and unconventional technologies to recover natural astaxanthin from microbial biomass.

Microorganisms such as bacteria, microalgae and fungi, are natural and rich sources of several valuable bioactive antioxidant's compounds, including carotenoids. Among the carotenoids with antioxidant properties, astaxanthin can be highlighted due to its pharmaceutical, feed, food, cosmetic and biotechnological applications. The best-known producers of astaxanthin are yeast and microalgae cells that biosynthesize this pigment intracellularly, requiring efficient and sustainable downstream procedures for its recovery. Conventional multi-step procedures usually involve the consumption of large amounts of volatile organic compounds (VOCs), which are regarded as toxic and hazardous chemicals. Considering these environmental issues, this review is focused on revealing the potential of unconventional extraction procedures [viz., Supercritical Fluid Extraction (SFE), Ultrasound-Assisted Extraction (UAE), Microwave-Assisted Extraction (MAE), High-Pressure Homogenization (HPH)] combined with alternative green solvents (biosolvents, eutectic solvents and ionic liquids) for the recovery of microbial-based astaxanthin from microalgae (such as Haematococcus pluvialis) and yeast (such as Phaffia rhodozyma) cells. The principal advances in the area, process bottlenecks, solvent selection and strategies to improve the recovery of microbial astaxanthin are emphasized. The promising recovery yields using these environmentally friendly procedures in lab-scale are good indications and directions for their effective use in biotechnological processes for the production of commercial feed and food ingredients like astaxanthin.

Advances and trends in biotechnological production of natural astaxanthin by Phaffia rhodozyma yeast.

Astaxanthin (AXT) is a natural xanthophyll with strong antioxidant, anticancer and antimicrobial activities, widely used in the food, feed, pharmaceutical and nutraceutical industries. So far, 95% of the AXT global market is produced by chemical synthesis, but growing customer preferences for natural products are currently changing the market for natural AXT, highlighting the production from microbially-based sources such as the yeast Phaffia rhodozyma. The AXT production by P. rhodozyma has been studied for a long time at a laboratory scale, but its use in industrial-scale processes is still very scarce. The optimization of growing conditions as well as an effective integration of upstream-downstream operations into P. rhodozyma-based AXT processes has not yet been fully achieved. With this critical review, we scrutinized the main approaches for producing AXT using P. rhodozyma strains, highlighting the impact of using conventional and non-conventional procedures for the extraction of AXT from yeast cells. In addition, we also pinpointed research directions, for example, the use of low-cost residues to improve the economic and environmental sustainability of the bioprocess, the use of environmentally/friendly and low-energetic integrative operations for the extraction and purification of AXT, as well as the need of further human clinical trials using yeast-based AXT.

Extracellular expression of Saccharomyces cerevisiae's L-asparaginase II in Pichia pastoris results in novel enzyme with better parameters.

L-asparaginase (ASNase) is an efficient inhibitor of tumor development, used in chemotherapy sessions against acute lymphoblastic leukemia (ALL) tumor cells; its use results in 80% complete remission of the disease in treated patients. Saccharomyces cerevisiae's L-asparaginase II (ScASNaseII) has a high potential to substitute bacteria ASNase in patients that developed hypersensitivity, but the endogenous production of it results in hypermannosylated immunogenic enzyme. Here we describe the genetic process to acquire the ScASNaseII expressed in the extracellular medium. Our strategy involved a fusion of mature sequence of protein codified by ASP3 (amino acids 26-362) with the secretion signal sequence of Pichia pastoris acid phosphatase enzyme; in addition, this DNA construction was integrated in P. pastoris Glycoswitch® strain genome, which has the cellular machinery to express and secrete high quantity of enzymes with humanized glycosylation. Our data show that the DNA construction and strain employed can express extracellular asparaginase with specific activity of 218.2 IU mg-1. The resultant enzyme is 40% more stable than commercially available Escherichia coli's ASNase (EcASNaseII) when incubated with human serum. In addition, ScASNaseII presents 50% lower cross-reaction with anti-ASNase antibody produced against EcASNaseII when compared with ASNase from Dickeya chrysanthemi.

Effects of Oxygen Transference on Protease Production by Rhodotorula mucilaginosa CBMAI 1528 in a Stirred Tank Bioreactor.

Microbial proteases, especially aspartic proteases, are an essential group of enzymes produced from different microorganisms. Microbial proteases have several applications, mainly in the food, beverage, cosmetic, and pharmaceutical industries, due to their efficiency in the processing and in the manufacturing stages. The yeast Rhodotorula mucilaginosa CBMAI 1528 was isolated from the Antarctic environment and was previously reported to have higher extracellular aspartic protease production. In addition, advances in the operational conditions of bioreactors for enzyme production are important to reduce the gap associated with scaling-up processes. This is the first study that evaluates the influence of oxygen transference (kLa) on the protease production of R. mucilaginosa yeast. To that end, batch cultures were created in a stirred tank bioreactor using Sabouraud dextrose broth at 25 °C for 72 h under kLa values from 18 to 135 h-1. The results show that kLa (121 h-1) obtained at 500 rpm and 1.5 vvm plays an important role in protease production (124.9 U/mL) and productivity (6.784 U/L.h) as well as biomass (10.4 g/L), µmax (0.14 h-1) and Yx/s (0.484 g/g). In conclusion, R. mucilaginosa showed high yield production in aerobic culture with the efficiency of protease expression and secretion influenced by kLa. In this sense, our results could be used for further industrial investment.

Helicobacter pyloril-asparaginase: a study of immunogenicity from an in silico approach.

Helicobacter pylori has become the causal agent of multiple forms of gastric disease worldwide, including gastric cancer. The enzyme l-asparaginase (ASNase) has been studied as a virulence factor. In this work, we performed an in silico investigation to characterize the immunological profile of H. pylori ASNase (HpASNase) to ascertain the possible implication of HpASNase immunogenicity in the H. pylori virulence mechanism. We applied a workflow based on bioinformatics tools, which, by calculating the relative frequency of immunogenic T-cell and B-cell epitopes, allowed us to predict the immunogenicity and allergenicity of HpASNase in silico. We also visualized the epitopes by mapping them into the native structure of the enzyme. We report for the first time the T-cell and B-cell epitope composition that contributes to the immunogenicity of this HpASNase, as well as the regions that could generate a hypersensitivity response in humans. ASNase from H. pylori resulted in highly immunogenic and allergenic. The high immunogenicity of HpASNase could imply the pathogenic mechanisms of H. pylori. This knowledge could be important for the development of new drugs against H. pylori infections. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03359-0.

Peg-Grafted Liposomes for L-Asparaginase Encapsulation.

L-asparaginase (ASNase) is an important biological drug used to treat Acute Lymphoblastic Leukemia (ALL). It catalyzes the hydrolysis of L-asparagine (Asn) in the bloodstream and, since ALL cells cannot synthesize Asn, protein synthesis is impaired leading to apoptosis. Despite its therapeutic importance, ASNase treatment is associated to side effects, mainly hypersensitivity and immunogenicity. Furthermore, degradation by plasma proteases and immunogenicity shortens the enzyme half-life. Encapsulation of ASNase in liposomes, nanostructures formed by the self-aggregation of phospholipids, is an attractive alternative to protect the enzyme from plasma proteases and enhance pharmacokinetics profile. In addition, PEGylation might prolong the in vivo circulation of liposomes owing to the spherical shielding conferred by the polyethylene (PEG) corona around the nanostructures. In this paper, ASNase was encapsulated in liposomal formulations composed by 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) containing or not different concentrations of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N [methoxy (polyethylene glycol)-2000] (DSPE-PEG). Nanostructures of approximately 142-202 nm of diameter and polydispersity index (PDI) of 0.069 to 0.190 were obtained and the vesicular shape confirmed by Transmission Electron Microscopy (TEM and cryo-TEM). The encapsulation efficiency (%EE) varied from 10% to 16%. All formulations presented activity in contact with ASNase substrate, indicating the liposomes permeability to Asn and/or enzyme adsorption at the nanostructures' surface; the highest activity was observed for DMPC/DSPE-PEG 10%. Finally, we investigated the activity against the Molt 4 leukemic cell line and found a lower IC50 for the DMPC/DSPE-PEG 10% formulation in comparison to the free enzyme, indicating our system could provide in vivo activity while protecting the enzyme from immune system recognition and proteases degradation.

A look into Phaffia rhodozyma biorefinery: From the recovery and fractionation of carotenoids, lipids and proteins to the sustainable manufacturing of biologically active bioplastics.

Carotenoids over-producing yeast has become a focus of interest of the biorefineries, in which the integration of the bioproduction with the following downstream processing units for the recovery and purification of carotenoids and other value-added byproducts is crucial to improve the sustainability and profitability of the overall bioprocess. Aiming the future implementation of Phaffia rhodozyma-based biorefineries, in this work, an integrative process for fractionation of intracellular compounds from P. rhodozyma biomass using non-hazardous bio-based solvents was developed. After one-extraction step, the total amount of astaxanthin, ß-carotene, lipids and proteins recovered was 63.11 µg/gDCW, 42.81 µg/gDCW, 53.75 mg/gDCW and 10.93 mg/g, respectively. The implementation of sequential back-extraction processes and integration with saponification and precipitation operations allowed the efficient fractionation and recovery (% w/w) of astaxanthin (∼72.5 %), ß-carotene âˆ¼90.17 %), proteins (21.04 %) and lipids (23.72 %). After fractionation, the manufacture of carotenoids-based products was demonstrated, through the mixture of carotenoids-rich extracts with bacterial cellulose to obtain biologically active bioplastics.

Structural and functional diversity of asparaginases: Overview and recommendations for a revised nomenclature.

Asparaginases (ASNases) are a large and structurally diverse group of enzymes ubiquitous amongst archaea, bacteria and eukaryotes, that catalyze hydrolysis of asparagine to aspartate and ammonia. Bacterial ASNases are important biopharmaceuticals for the treatment of acute lymphoblastic leukemia, although some patients experience adverse allergic side effects during treatment with these protein therapeutics. ASNases are currently divided into three families: plant-type ASNases, Rhizobium etli-type ASNases and bacterial-type ASNases. This system is outdated as both bacterial-type and plant-type families also include archaeal, bacterial and eukaryotic enzymes, each with their own distinct characteristics. Herein, phylogenetic studies allied to tertiary structural analyses are described with the aim of proposing a revised and more robust classification system that considers the biochemical diversity of ASNases. Accordingly, based on distinct peptide domains, phylogenetic data, structural analysis and functional characteristics, we recommend that ASNases now be divided into three new distinct classes containing subgroups according to structural and functional aspects. Using this new classification scheme, 25 ASNases were identified as candidates for future new lead discovery.