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.

Improving the production of recombinant L-Asparaginase-II in Escherichia coli by co-expressing catabolite repressor activator (cra) gene.

Identification of a single genetic target for microbial strain improvement is difficult due to the complexity of the genetic regulatory network. Hence, a more practical approach is to identify bottlenecks in the regulatory networks that control critical metabolic pathways. The present work focuses on enhancing cellular physiology by increasing the metabolic flux through the central carbon metabolic pathway. Global regulator cra (catabolite repressor activator), a DNA-binding transcriptional dual regulator was selected for the study as it controls the expression of a large number of operons that modulate central carbon metabolism. To upregulate the activity of central carbon metabolism, the cra gene was co-expressed using a plasmid-based system. Co-expression of cra led to a 17% increase in the production of model recombinant protein L-Asparaginase-II. A pulse addition of 0.36% of glycerol every two hours post-induction, further increased the production of L-Asparaginase-II by 35% as compared to the control strain expressing only recombinant protein. This work exemplifies that upregulating the activity of central carbon metabolism by tuning the expression of regulatory genes like cra can relieve the host from cellular stress and thereby promote the growth as well as expression of recombinant hosts.

A novel type IIb L-asparaginase from Latilactobacillus sakei LK-145: characterization and application.

We succeeded in homogeneously expressing and purifying L-asparaginase from Latilactobacillus sakei LK-145 (Ls-Asn1) and its mutated enzymes C196S, C264S, C290S, C196S/C264S, C196S/C290S, C264S/C290S, and C196S/C264S/C290S-Ls-Asn1. Enzymological studies using purified enzymes revealed that all cysteine residues of Ls-Asn1 were found to affect the catalytic activity of Ls-Asn1 to varying degrees. The mutation of Cys196 did not affect the specific activity, but the mutation of Cys264, even a single mutation, significantly decreased the specific activity. Furthermore, C264S/C290S- and C196S/C264S/C290S-Ls-Asn1 almost completely lost their activity, suggesting that C290 cooperates with C264 to influence the catalytic activity of Ls-Asn1. The detailed enzymatic properties of three single-mutated enzymes (C196S, C264S, and C290S-Ls-Asn1) were investigated for comparison with Ls-Asn1. We found that only C196S-Ls-Asn1 has almost the same enzymatic properties as that of Ls-Asn1 except for its increased stability for thermal, pH, and the metals NaCl, KCl, CaCl2, and FeCl2. We measured the growth inhibitory effect of Ls-Asn1 and C196S-Ls-Asn1 on Jurkat cells, a human T-cell acute lymphoblastic leukemia cell line, using L-asparaginase from Escherichia coli K-12 as a reference. Only C196S-Ls-Asn1 effectively and selectively inhibited the growth of Jurkat T-cell leukemia, which suggested that it exhibited antileukemic activity. Furthermore, based on alignment, phylogenetic tree analysis, and structural modeling, we also proposed that Ls-Asn1 is a so-called "Type IIb" novel type of asparaginase that is distinct from previously reported type I or type II asparaginases. Based on the above results, Ls-Asn1 is expected to be useful as a new leukemia therapeutic agent.

L-Asparaginase Conjugates from the Hyperthermophilic Archaea Thermococcus sibiricus with Improved Biocatalytic Properties.

This study investigated the effect of polycationic and uncharged polymers (and oligomers) on the catalytic parameters and thermostability of L-asparaginase from Thermococcus sibiricus (TsA). This enzyme has potential applications in the food industry to decrease the formation of carcinogenic acrylamide during the processing of carbohydrate-containing products. Conjugation with the polyamines polyethylenimine and spermine (PEI and Spm) or polyethylene glycol (PEG) did not significantly affect the secondary structure of the enzyme. PEG contributes to the stabilization of the dimeric form of TsA, as shown by HPLC. Furthermore, neither polyamines nor PEG significantly affected the binding of the L-Asn substrate to TsA. The conjugates showed greater maximum activity at pH 7.5 and 85 °C, 10-50% more than for native TsA. The pH optima for both TsA-PEI and TsA-Spm conjugates were shifted to lower pH ranges from pH 10 (for the native enzyme) to pH 8.0. Additionally, the TsA-Spm conjugate exhibited the highest activity at pH 6.5-9.0 among all the samples. Furthermore, the temperature optimum for activity at pH 7.5 shifted from 90-95 °C to 80-85 °C for the conjugates. The thermal inactivation mechanism of TsA-PEG appeared to change, and no aggregation was observed in contrast to that of the native enzyme. This was visually confirmed and supported by the analysis of the CD spectra, which remained almost unchanged after heating the conjugate solution. These results suggest that TsA-PEG may be a more stable form of TsA, making it a potentially more suitable option for industrial use.

Application of sequential design for enhanced L-asparaginase synthesis from Ganoderma australe GPC191.

With an increasing demand for L-asparaginase in pharmaceutical and food sectors for its cytostatic and acrylamide-reducing qualities, there's a need to discover novel, highly productive enzyme sources with improved pharmacokinetic profiles. Keeping this in mind, the present study aimed at maximizing the potential of Ganoderma australe GPC191 to produce L-asparaginase by fermentation medium optimization using statistical validation. Of the 11 physicochemical parameters evaluated under submerged fermentation conditions through one-factor-at-a-time approach and Plackett-Burman design, only four parameters (inoculum load, L-asparagine, soybean meal, and initial pH) influenced L-asparaginase production, significantly (p < 0.001). The optimal levels and interaction effects of these on the overall production were further evaluated by the central composite rotatable design of response surface methodology. Post-optimization, 27.34 U/mL was predicted as the maximum activity at pH 7 with 5n inoculum load and 15 g/L each of L-asparagine and soybean meal. Experimental validation yielded an activity of 28.52 U/mL, indicating an overall 18.17-fold increase from the unoptimized stage. To our knowledge, this is the first report signifying the L-asparaginase production aptitude of G. australe with sequential statistical validation using agricultural waste, which can serve as a model to enhance its yields, offering a sustainable and cost-effective solution for industrial application.

Synergistic effects of ternary mixture formulation and process parameters optimization in a sequential approach for enhanced L-asparaginase production using agro-industrial wastes.

A novel ternary mixture of inexpensive and nutrient-rich agro-substrates comprising groundnut de-oiled cake, corn gluten meal, and soybean meal has been explored to enhance the L-asparaginase production in solid-state fermentation. To achieve the aim, a hybrid strategy was implemented by utilizing a combination of a mixture design and artificial neural networks. The study initiated with the judicious selection of the agro-substrates based on their low C/N content in comparison to the control using the CHNS elemental analysis. The mixture composition of soybean meal (49.0%), groundnut de-oiled cake (31.5%), and corn gluten meal (19.5%) were found optimum using the simplex lattice mixture design. The agro-industrial substrates mix revealed synergistic effects on the L-asparaginase production than either of the substrates alone. The maximum L-asparaginase activity of 141.45 ± 5.24 IU/gds was observed under the physical process conditions of 70% moisture content, autoclaving period of 30 min and 6.0 pH by adopting the machine learning-derived artificial neural network (ANN) methodology. The ANN modeling showed excellent prediction ability with a low mean squared error of 0.7, a low root mean squared error of 0.84, and a high value of 0.99 for regression coefficient. Moisture content (%) was assessed to be the most sensitive process parameter in the global sensitivity analysis. The net outcome from the two sequential optimization designs is the selection of the ideal mixture composition followed by the optimum physical process parameters. The application of the enzyme demonstrated significant cytotoxicity against leukemia cell line and therefore exhibited an anti-cancer effect. The present study reports a novel mixture combination and methodology that can be used to lower the cost and enhance the production of L-asparaginase using an agro-industrial substrate mixture.

Heterologous expression of fungal L-asparaginase: a systematic review.

Aim: To review the available literature about heterologous expression of fungal L-asparaginase (L-ASNase). Materials & methods: A search was conducted across PubMed, Science Direct, Scopus and Web of Science databases; 4172 citations were identified and seven articles were selected. Results: The results showed that heterologous expression of fungal L-ASNase was performed mostly in bacterial expression systems, except for a study that expressed L-ASNase in a yeast system. Only three publications reported the purification and characterization of the enzyme. Conclusion: The information reported in this systematic review can contribute significantly to the recognition of the importance of biotechnological techniques for L-ASNase production.

Asparaginase is a common treatment for the most common type of leukemia in children. These treatments generally use asparaginase sourced from bacteria. Some people can experience bad reactions to these treatments. One way that has been explored to avoid this is to use asparaginase sourced from fungi because they are more similar to humans. However, fungi produce less asparaginase than bacteria. This review looks into ways that the production of fungal asparaginases can be made more productive.

Apoptosis and cell cycle arrest of leukemic cells by a robust and stable L-asparaginase from Pseudomonas sp. PCH199.

Biomolecules obtained from microorganisms living in extreme environments possess properties that have pharmacokinetic advantages. Enzyme assay revealed recombinant L-ASNase, an extremozyme from Pseudomonas sp. PCH199 is to be highly stable with 90 % activity (200 h) at 37 °C. The stability of the enzyme in human serum (50 % activity maintained in 63 h) reveals high therapeutic potential with less dosage. The enzyme exhibited cytotoxicity to K562 blood cancer cell lines with IC50 of 0.37 U/mL without affecting the IEC-6 normal epithelial cell line. Due to the depletion of L-asparagine, K562 cells experience nutritional stress that results in the abruption of metabolic processes and eventually leads to apoptosis. Comparative studies on MCF-7 cells also revealed the same fate. Due to nutritional stress induced by L-ASNase treatment, mitochondrial membrane potential was lost, and reactive oxygen species were increased to 48 % (K562) and 21 % (MCF-7) as indicated by flow cytometric analysis. DAPI staining with prominent nuclear morphological changes visualized under the fluorescent microscope confirmed apoptosis in both cancer cells. Treatment increases pro-apoptotic Bax protein, and eventually, the cell cycle is arrested at the G2/M phase in both cell lines. Therefore, the current study paves the way for PCH199 L-ASNase to be considered a potential chemotherapeutic agent for treating acute lymphoblastic leukemia.

Characterization and applications of glutaminase free L-asparaginase from indigenous Bacillus halotolerans ASN9.

L-asparaginase (L-ASNase) is a versatile anticancer and acrylamide reduction enzyme predominantly used in medical and food industries. However, the high specificity of L-asparaginase formulations for glutamine, low thermostability, and blood clearance are the major disadvantages. Present study describes production, characterization, and applications of glutaminase free extracellular L-asparaginase from indigenous Bacillus halotolerans ASN9 isolated from soil sample. L-asparaginase production was optimized in M9 medium (containing 0.2% sucrose and 1% L-asparagine) that yielded maximum L-ASNase with a specific activity of 256 U mg-1 at pH 6 and 37°C. L-asparaginase was purified through acetone precipitation and Sephadex G-100 column, yielding 48.9 and 24% recovery, respectively. Enzyme kinetics revealed a Vmax of 466 mM min-1 and Km of 0.097 mM. Purified L-ASNase showed no activity against glutamine. The purified glutaminase free L-ASNase has a molecular mass of 60 kDa and an optimum specific activity of 3083 U mg-1 at pH 7 and 37°C. The enzyme retains its activity and stability over a wide range of pH and temperature, in the presence of selected protein inhibitors (SDS, ß-mercaptoethanol), CoCl2, KCl, and NaCl. The enzyme also exhibited antioxidant activity against DPPH radical (IC50 value 70.7 µg mL-1) and anticancer activity against U87 human malignant glioma (IC50 55 µg mL-1) and Huh7 human hepatocellular carcinoma (IC50 37 µg mL-1) cell lines. Normal human embryonic kidney cells (HEK293) had greater than 80% cell viability with purified L-ASNase indicating its least cytotoxicity against normal cells. The present work identified potent glutaminase free L-ASNase from B. halotolerans ASN9 that performs well in a wide range of environmental conditions indicating its suitability for various commercial applications.

Utility of ASNS gene methylation evaluated with the HPLC method as a pharmacogenomic biomarker to predict asparaginase sensitivity in BCP-ALL.

Asparaginase is an important agent for the treatment of acute lymphoblastic leukaemia (ALL), but it is occasionally associated with severe adverse events. Thus, for safer and more efficacious therapy, a clinical biomarker predicting asparaginase sensitivity is highly anticipated. Asparaginase depletes serum asparagine by deaminating asparagine into aspartic acid, and ALL cells are thought to be sensitive to asparaginase due to reduced asparagine synthetase (ASNS) activity. We have recently shown that allele-specific methylation of the ASNS gene is highly involved in asparaginase sensitivity in B-precursor ALL (BCP-ALL) by using next-generation sequence (NGS) analysis of bisulphite PCR products of the genomic DNA. Here, we sought to confirm the utility of methylation status of the ASNS gene evaluated with high-performance liquid chromatography (HPLC) analysis of bisulphite PCR products for future clinical applications. In the global methylation status of 23 CpG sites at the boundary region of promoter and exon 1 of the ASNS gene, a strong positive correlation was confirmed between the mean percent methylation evaluated with the HPLC method and that with the NGS method in 79 BCP-ALL cell lines (R2 = 0.85, p = 1.3 × 10-33) and in 63 BCP-ALL clinical samples (R2 = 0.84, p = 5.0 × 10-26). Moreover, methylation status of the ASNS gene evaluated with the HPLC method was significantly associated with in vitro asparaginase sensitivities as well as gene and protein expression levels of ASNS. These observations indicated that the ASNS gene methylation status evaluated with the HPLC method is a reliable biomarker for predicting the asparaginase sensitivity of BCP-ALL.

Characterization of the Type 2 L-Asparaginase Gene in Thermohalophilic Bacterial from Wawolesea Hot Springs, Southeast Sulawesi, Indonesia.

<b>Background and Objective:</b> Type 2 L-asparaginase enzyme can be used as a cancer therapy agent and prevent acrylamide formation in food products. Enzymes produced by thermohalophilic bacteria can provide high activity at high temperatures so they are needed on an industrial scale. Hence, this study aims to determine the characteristics of the gene encoding type 2 L-asparaginase enzyme in the thermohalophilic bacterial isolate CAT3.4. <b>Materials and Methods:</b> This research is a type of exploratory research. The characteristics of the gene encoding type 2 L-asparaginase were determined using the PCR technique using the primer pairs AsnBac2-F2 (5'-CTCACGGGAATCTCCATAACTC-3') and AsnBac2-R2 (5'CAGCGATGTAACAGACAGCATC-3'). The characterization process was carried out in stages: Isolation of genomic DNA using a modified alkali-lysis method, nucleotide and protein similarity analysis using BLASTn analysis on the NCBI website, construction of a phylogenetic tree using the MEGAX program, restriction enzyme mapping and amino acid analysis using the Bioedit program. <b>Results:</b> The characterization results showed that the PCR product has a size of 1594 bp with a CDS of 1128 bp, has a similarity value of 100% with <i>Bacillus subtilis</i>, has seven restriction enzymes as molecular markers for the type 2 L-asparaginase gene at the species level: <i>Bsr</i>GI, <i>Dra</i>I, <i>Eco</i>RV, <i>Hind</i>III, <i>Hpy</i>CH4IV , <i>Ssp</i>I and <i>Tai</i>I, have dominant hydrophilic regions and are in the same subclass as <i>Bacillus subtilis</i> strain GOT9. <b>Conclusion:</b> The target gene was similar to the gene encoding type 2 L-asparaginase from <i>Bacillus subtilis</i> with a max identity of 98.85%, query coverage value of 100% and E-value of 0.

Engineering and Expression Strategies for Optimization of L-Asparaginase Development and Production.

Genetic engineering for heterologous expression has advanced in recent years. Model systems such as Escherichia coli, Bacillus subtilis and Pichia pastoris are often used as host microorganisms for the enzymatic production of L-asparaginase, an enzyme widely used in the clinic for the treatment of leukemia and in bakeries for the reduction of acrylamide. Newly developed recombinant L-asparaginase (L-ASNase) may have a low affinity for asparagine, reduced catalytic activity, low stability, and increased glutaminase activity or immunogenicity. Some successful commercial preparations of L-ASNase are now available. Therefore, obtaining novel L-ASNases with improved properties suitable for food or clinical applications remains a challenge. The combination of rational design and/or directed evolution and heterologous expression has been used to create enzymes with desired characteristics. Computer design, combined with other methods, could make it possible to generate mutant libraries of novel L-ASNases without costly and time-consuming efforts. In this review, we summarize the strategies and approaches for obtaining and developing L-ASNase with improved properties.

Response surface methodology based optimized production, purification, and characterization of L-asparaginase from Fusarium foetens.

L-asparaginase is used as one of the prime chemotherapeutic agents to treat acute lymphoblastic leukemia. L-asparaginase obtained from bacteria exhibits hypersensitive reactions including various side effects. The present work aimed to optimize growth parameters for maximum production of L-asparaginase by Fusarium foetens through response surface methodology, its purification, and characterization. The optimization of L-asparaginase production by Fusarium foetens was initially done through a one-factor-at-a-time method. L-asparaginase production was further optimized using a central composite design based response surface methodology. The maximum L-asparaginase activity of 12.83 IU/ml was obtained under the following growth conditions; temperature-27.5 °C, pH-8, inoculum concentration-1.5 × 106 spores/ml, and incubation period-7 days. In comparison with the unoptimized growth conditions (4.58 IU/ml), the optimization led to a 2.65-fold increase in the L-asparaginase activity. The L-asparaginase from Fusarium foetens was purified 15.60-fold, with a yield of 39.89% using DEAE-cellulose column chromatography. After purification, the L-asparaginase activity was determined to be 127.26 IU/ml and the specific activity was found to be 231.38 IU/mg. The molecular mass was estimated to be approximately 37 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme showed optimum activity at pH 5, and a temperature of 40 °C. The enzyme showed 100% specificity towards L-asparagine and no activity towards L-glutamine. Its activity was enhanced by Mn2+, Fe2+, and Mg2, while it was inhibited by ß-mercaptoethanol and EDTA. The Km and Vmax of the purified L-asparaginase were found to be 23.82 mM and 210.3 IU/ml respectively. The results suggest that Fusarium foetens could be a potent candidate for the bioprocessing of L-asparaginase at a large scale.

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.

Endophytic Fungi as a Promising Source of Anticancer L-Asparaginase: A Review.

L-asparaginase is a tetrameric enzyme from the amidohydrolases family, that catalyzes the breakdown of L-asparagine into L-aspartic acid and ammonia. Since its discovery as an anticancer drug, it is used as one of the prime chemotherapeutic agents to treat acute lymphoblastic leukemia. Apart from its use in the biopharmaceutical industry, it is also used to reduce the formation of a carcinogenic substance called acrylamide in fried, baked, and roasted foods. L-asparaginase is derived from many organisms including plants, bacteria, fungi, and actinomycetes. Currently, L-asparaginase preparations from Escherichia coli and Erwinia chrysanthemi are used in the clinical treatment of acute lymphoblastic leukemia. However, they are associated with low yield and immunogenicity problems. At this juncture, endophytic fungi from medicinal plants have gained much attention as they have several advantages over the available bacterial preparations. Many medicinal plants have been screened for L-asparaginase producing endophytic fungi and several studies have reported potent L-asparaginase producing strains. This review provides insights into fungal endophytes from medicinal plants and their significance as probable alternatives for bacterial L-asparaginase.

L-asparaginase activity and anti-L-asparaginase antibody as biomarkers in estimating PEG-asp-related anaphylaxis risk in childhood acute lymphoblastic leukemia patients

Background: L-Asparaginase (L-asp), the unconjugated form of polyethylene glycol-conjugated L-asparaginase (PEG-asp), regulates T cell stimulation, antibody production, and lysosomal protease activity to mediate PEG-asp-related anaphylaxis. This study aimed to investigate the relation of L-asp activity and anti-L-asp antibody with anaphylaxis risk and non-anaphylaxis adverse reaction risk in childhood acute lymphoblastic leukemia (ALL) patients who underwent PEG-asp contained therapy. Methods: In total, 170 childhood ALL patients underwent PEG-asp-contained treatment and their L-asp activity and anti-L-asp antibody were detected on the 7th day after treatment initiation. Results: There were 27 (15.9%) patients who had PEG-asp-related adverse reaction: 17 (10.0%) patients experienced PEG-asp-related anaphylaxis and 14 (8.2%) patients experienced PEG- asp-related non-anaphylaxis adverse reaction. Moreover, L-asp activity was negatively related to anti-L-asp antibody in childhood ALL patients (P<0.001). Elevated L-asp activity was associated with the absence of PEG-asp-related anaphylaxis (P<0.001), PEG-asp-related non-anaphylaxis adverse reaction (P=0.004), and PEG-asp-related adverse reaction (P<0.001). However, the anti- L-asp antibody displayed opposite trend similar to L-asp activity. Receiver operating characteristic (ROC) curve analyses exhibited L-asp activity and anti-L-asp antibody exhibited superior predictive values in estimating PEG-asp-related anaphylaxis risk with area under curve (AUC) of 0.955 and 0.905, respectively compared to PEG-asp-related non-anaphylaxis adverse reaction risk with AUC of 0.730 and 0.675, respectively. Besides, patients with de novo disease, higher risk stratification, and allergic history showed trends linked with PEG-asp-related anaphylaxis risk. Conclusion: The monitoring of L-asp activity and anti-L-asp antibody maybe useful for early estimation and prevention of PEG-asp-related anaphylaxis in childhood ALL management (AU)

Comparative transcriptomics of aphid species that diverged > 22 MYA reveals genes that are important for the maintenance of their symbiosis.

Most plant-sap feeding insects have obligate relationships with maternally transmitted bacteria. Aphids require their nutritional endosymbiont, Buchnera aphidicola, for the production of essential amino acids. Such endosymbionts are harbored inside of specialized insect cells called bacteriocytes. Here, we use comparative transcriptomics of bacteriocytes between two recently diverged aphid species, Myzus persicae and Acyrthosiphon pisum, to identify key genes that are important for the maintenance of their nutritional mutualism. The majority of genes with conserved expression profiles in M. persicae and A. pisum are for orthologs previously identified in A. pisum to be important for the symbiosis. However, asparaginase which produces aspartate from asparagine was significantly up-regulated only in A. pisum bacteriocytes, potentially because Buchnera of M. persicae encodes its own asparaginase enzyme unlike Buchnera of A. pisum resulting in Buchnera of A. pisum to be dependent on its aphid host for aspartate. One-to-one orthologs that explained the most amount of variation for bacteriocyte specific mRNA expression for both species includes a collaborative gene for methionine biosynthesis, multiple transporters, a horizontally transmitted gene, and secreted proteins. Finally, we highlight species-specific gene clusters which may contribute to host adaptations and/or accommodations in gene regulation to changes in the symbiont or the symbiosis.

Characterization of L-asparaginase from Streptomyces koyangensis SK4 with acrylamide-minimizing potential in potato chips.

Microbial L-asparaginase is well known for its application in food industries to reduce acrylamide content in fried starchy food. L-asparaginase produced by Arctic actinomycetes Streptomyces koyangensis SK4 was purified and studied for biochemical characterization. The L-asparaginase was purified with a yield of 15.49% and final specific activity of 179.77 IU/mg of protein. The enzyme exhibited a molecular weight of 43 kDa. The optimum pH and temperature for maximum activity of the purified enzyme were 8.5 °C and 40 °C, respectively. The enzyme expressed maximum activity at an incubation period of 30 min and a substrate concentration of 0.06 M. The enzyme has a low Km value of 0.041 M and excellent substrate specificity toward L-asparagine. The enzyme activity was inhibited by metal ions Ba2+ and Hg2+, while Mn2+ and Mg2+ enhanced the activity. The study evaluated the acrylamide reduction potential of L-asparaginase from Streptomyces koyangensis SK4 in potato chips. The blanching plus L-asparaginase treatment of potato slices resulted in a 50% reduction in acrylamide content. The study illustrated an effective acrylamide reduction strategy in potato chips using L-asparaginase from a psychrophilic actinomycete. Besides the acrylamide reduction potential, L-asparaginase from Streptomyces koyangensis SK4 also did not exhibit any glutaminase or urease activity which is an outstanding feature of L-asparaginase to be used as a chemotherapeutic agent.

L-asparaginase immobilization in supramolecular nanogels of PEG-grafted poly HPMA and bis(α-cyclodextrin) to enhance pharmacokinetics and lower enzyme antigenicity.

L-asparaginase (ASNase) enzyme has limited therapeutic use due to its poor pharmacokinetics and immunogenicity. To overcome these obstacles, we immobilized ASNase in biocompatible poly hydroxypropyl methacrylamide (P(HPMA))-based nanogels simply formed through the host-guest inclusion complex of ASNase-conjugated random copolymer of HPMA and polyethylene glycol (PEG) acrylate (P(HPMA-MPEGA)) and α-cyclodextrin dimer (bisCD) using cystamine as a linker. The effects of bisCD and polymer concentrations on particle size, gelation time, and recovery of enzyme activity were investigated. The ASNase-conjugated bisCD nanogels were discrete, homogeneous, and spherical with a mean projected diameter of 148 ± 41 nm. ASNase immobilized in the bisCD nanogels caused cytotoxicity on HL-60 cell line with IC50 of 3 IU/ml. In-vivo rat study revealed that the immobilized ASNase reduced the enzyme antigenicity and resulted in 8.1 folds longer circulation half-life than the native enzyme. Conclusively, immobilization of ASNase in P(HPMA-MPEGA) and bisCD supramolecular nanogels could enhance the therapeutic value of ASNase in cancer chemotherapy.

Asparagine and Glutamine Deprivation Alters Ionizing Radiation Response, Migration and Adhesion of a p53null Colorectal Cancer Cell Line.

Colorectal cancer (CRC) is the most prominent form of colon cancer for both incidence (38.7 per 100,000 people) and mortality (13.9 per 100,000 people). CRC's poor response to standard therapies is linked to its high heterogeneity and complex genetic background. Dysregulation or depletion of the tumor suppressor p53 is involved in CRC transformation and its capability to escape therapy, with p53null cancer subtypes known, in fact, to have a poor prognosis. In such a context, new therapeutic approaches aimed at reducing CRC proliferation must be investigated. In clinical practice, CRC chemotherapy is often combined with radiation therapy with the aim of blocking the expansion of the tumor mass or removing residual cancer cells, though contemporary targeting of amino acid metabolism has not yet been explored. In the present study, we used the p53null Caco-2 model cell line to evaluate the effect of a possible combination of radiation and L-Asparaginase (L-ASNase), a protein drug that blocks cancer proliferation by impairing asparagine and glutamine extracellular supply. When L-ASNase was administered immediately after IR, we observed a reduced proliferative capability, a delay in DNA-damage response and a reduced capability to adhere and migrate. Our data suggest that a correctly timed combination of X-rays and L-ASNase treatment could represent an advantage in CRC therapy.