The effect of protein-glutaminase from Chryseobacterium proteolyticum on physicochemical and functional properties of high-temperature soybean meal protein.

Protein-glutaminase (EC 3.5.1.44, PG) can significantly improve the functional properties of food proteins. However, the low yield of PG has limited industrial applications. Results showed that 0.02 % tea saponin could increase the PG yield by 18.93 %. The transcription level of the PG gene was significantly enhanced, which promoted the extracellular secretion of PG through an increase in membrane permeability. On this basis, PG was used to modify high-temperature soybean meal protein (HSMP) due to its poor properties. In this study, the deamidation degree (DD) of PG-modified HSMP was optimized to 58.61 % by the response surface method. HSMP with different DD was prepared and its physicochemical and functional properties were studied. After PG treatment, the intermolecular repulsive force of HSMP increased, the particle size distribution became uniform, and the solution system was more stable. In addition, the surface morphology of HSMP gradually became loose and porous. The solubility of HSMP significantly improved, reaching 11.34 times that of untreated HSMP at pH 5.00. Meanwhile, the emulsifying and foaming capacity of HSMP significantly improved, but the foaming stability was reduced.

Effects of protein-glutaminase on the properties of glutinous rice flour, paste, and gel food: Based on the interactions between the deamidated protein and starch.

In the present study, protein-glutaminase (PG) from Chryseobacterium proteolyticum was applied to improve the processing properties of glutinous rice flour (GRF). After PG modification, the degree of deamidation of glutinous rice protein (GRP) reached 13.6% at 2.0 h, with smaller particle size and decreased zeta potential. The interaction of GRP with starch in PG-modified GRF (PM-GRF) was changed, exhibiting in protein aggregates decreasing and exposure of starch on the surface of GRF. Compared with unmodified GRF (UM-GRF), the solubility and turbidity of PM-GRF were both increased. The rheological properties reflected that the viscosity of PM-GRF paste was increased, and the freeze-thaw stability was also enhanced. Moreover, the textural characteristics showed that the hardness of PM-GRF balls remarkably reduced and the springiness increased. These results indicate that deamidation by PG could be an efficient method for improving characteristics of GRP and GRF.

Molecular expression, purification and structural characterization of recombinant L-Glutaminase from Streptomyces roseolus.

The present research reports the anti-cancer potential of recombinant L-Glutaminase from Streptomyces roseolus. L-Glutaminase gene was synthesized by codon-optimization, cloned and successfully expressed in E. coli BL21 (DE3). Affinity purified recombinant L-Glutaminase revealed a molecular mass of 32 kDa. Purified recombinant L-Glutaminase revealed stability at pH 7.0-8.0 with optimum activity at 70 °C further indicating its thermostable nature based on thermodynamic characterization. Recombinant L-Glutaminase exhibited profound stability in the presence of several biochemical parameters and demonstrated its metalloenzyme nature and was also found to be highly specific towards favorable substrate (l-Glutamine) based on kinetics. It demonstrated antioxidant property and pronounced cytotoxic effect against breast cancer (MCF-7 cell lines) in a dose dependent behavior with IC50 of 40.68 µg/mL. Matrix-assisted laser desorption ionization-time of flight-mass spectroscopy (MALDI-TOF-MS) analysis of desired mass peaks ascertained the recombinant L-Glutaminase identity. N-terminal amino acid sequence characterization through Edman degradation revealed highest resemblance for L-glutaminase within the Streptomyces sp. family. The purified protein was characterized structurally and functionally by employing spectroscopic methods like Raman, circular dichroism and nuclear magnetic resonance. The thermostability was assessed by thermogravimetric analysis. The outcomes of the study, suggests the promising application of recombinant L-Glutaminase as targeted therapeutic candidate for breast cancer.

Discovery and mechanistic analysis of a novel source protein glutaminase PG5 and its potential application.

In this study, we successfully obtained a novel source protein glutaminase PG5 with specific activity of 10.4 U/mg, good tolerance and broad substrate profile through big data retrieval. Structural analysis and site-directed mutagenesis revealed that the catalytic pocket of Mature-PG5 contained a large number of aromatic amino acids and hydrophobic amino acids, and that Ser72 greatly affects the properties of the catalytic pocket and the affinity of PG5 for the substrate. In addition, molecular dynamics analysis revealed that the opening and closing between amino acid residues Gly65 and Thr66 with Cys164 at the catalytic cleft could affect substrate binding and product release. In addition, PG5 effectively improved the solubility of fish myofibrillar proteins under low-salt conditions while enhancing their foaming and emulsification properties. This study offers valuable insights into the catalytic mechanism of PG5, which will contribute to its future directed evolution and application in the food industry.

Development of a Universal One-Step Purification and Activation Method to Engineer Protein-Glutaminase through Rational Design.

Cytotoxic enzymes often exist as zymogens containing prodomains to keep them in an inactive state. Protein-glutaminase (PG), which can enhance various functional characteristics of food proteins, is an enzyme containing pro-PG and mature-PG (mPG). However, poor activity and stability limit its application while tedious purification and activation steps limit its high-throughput engineering. Here, based on structural analysis, we replaced the linker sequence between pro-PG and mPG with the HRV3C protease recognition sequence and then coexpressed it with HRV3C protease in Escherichia coli to develop an efficient one-step purification and activation method for PG. We then used this method to obtain several mutants designed by a combination of computer-aided approach and beneficial point mutations. The specific activity (131.6 U/mg) of the best variant D1 was 4.14-fold that of the wild type, and t1/2 and T5010 increased by 13 min and 7 °C, respectively. D1 could effectively improve the solubility and emulsification of wheat proteins, more than twice the effect of the wild type. We also discussed the mechanism underlying the improved properties of D1. In summary, we not only provide a universal one-step purification and activation method to facilitate zymogen engineering but also obtain an excellent PG mutant.

Molecular mechanism of glutaminase activation through filamentation and the role of filaments in mitophagy protection.

Glutaminase (GLS), which deaminates glutamine to form glutamate, is a mitochondrial tetrameric protein complex. Although inorganic phosphate (Pi) is known to promote GLS filamentation and activation, the molecular basis of this mechanism is unknown. Here we aimed to determine the molecular mechanism of Pi-induced mouse GLS filamentation and its impact on mitochondrial physiology. Single-particle cryogenic electron microscopy revealed an allosteric mechanism in which Pi binding at the tetramer interface and the activation loop is coupled to direct nucleophile activation at the active site. The active conformation is prone to enzyme filamentation. Notably, human GLS filaments form inside tubulated mitochondria following glutamine withdrawal, as shown by in situ cryo-electron tomography of cells thinned by cryo-focused ion beam milling. Mitochondria with GLS filaments exhibit increased protection from mitophagy. We reveal roles of filamentous GLS in mitochondrial morphology and recycling.

Enhanced protein glutaminase production from Chryseobacterium proteolyticum combining physico-chemical mutagenesis and resistance screening and its application to soybean protein isolates.

BACKGROUND: Protein glutaminase (PG) is a novel protein modification biotechnology that is increasingly being used in the food industry. However, the current level of fermentation of PG-producing strains still does not meet the requirements of industrial production. To obtain the mutant strains with high PG production, the atmospheric and room temperature plasma (ARTP) combined with LiCl chemical mutagen were used in mutagenesis of a PG producing Chryseobacterium proteolyticum 1003. RESULTS: A mutant strain (WG15) was successfully obtained based on malonic acid resistance screening after compound mutagenesis of the starting strain C. proteolyticum 1003 using ARTP with LiCl, and it was confirmed to be genetically stable in PG synthesis after 15 generations. The protein glutaminase production of WG15 was 2.91 U mL-1 after optimization of fermentation conditions, which is 48.69% higher than the original strain C. proteolyticum 1003. The PG obtained from fermentation showed good activities in deamidation of soy protein isolate. The solubility and foaming properties of the PG-treated soy protein isolate were significantly increased by 36.50% and 10.03%, respectively, when PG was added at the amount of 100 U mL-1 . In addition, the emulsifying activity and emulsion stability of the treated soy protein isolate were improved by 12.44% and 10.34%, respectively, on the addition of 10 U mL-1 PG. The secondary structure of the soy protein isolate changed after PG treatment, with an increased proportion of glutamate. CONCLUSION: The results of the present study indicate that the PG produced by this mutant strain could improve the functional properties of soybean protein isolate and the C. proteolyticum mutant WG15 has great potential in food industry. © 2023 Society of Chemical Industry.

In vivo stabilization of a less toxic asparaginase variant leads to a durable antitumor response in acute leukemia.

Asparagine is a non-essential amino acid since it can either be taken up via the diet or synthesized by asparagine synthetase. Acute lymphoblastic leukemia (ALL) cells do not express asparagine synthetase or express it only minimally, which makes them completely dependent on extracellular asparagine for their growth and survival. This dependency makes ALL cells vulnerable to treatment with L-asparaginase, an enzyme that hydrolyzes asparagine. To date, all clinically approved L-asparaginases have significant L-glutaminase co-activity, associated with non-immune related toxic side effects observed during therapy. Therefore, reduction of L-glutaminase co-activity with concomitant maintenance of its anticancer L-asparaginase effect may effectively improve the tolerability of this unique drug. Previously, we designed a new alternative variant of Erwinia chrysanthemi (ErA; Erwinaze) with decreased L-glutaminase co-activity, while maintaining its L-asparaginase activity, by the introduction of three key mutations around the active site (ErA-TM). However, Erwinaze and our ErA-TM variant have very short half-lives in vivo. Here, we show that the fusion of ErA-TM with an albumin binding domain (ABD)-tag significantly increases its in vivo persistence. In addition, we evaluated the in vivo therapeutic efficacy of ABD-ErA-TM in a B-ALL xenograft model of SUP-B15. Our results show a comparable long-lasting durable antileukemic effect between the standard-of-care pegylated-asparaginase and ABD-ErA-TM L-asparaginase, but with fewer co-glutaminase-related acute side effects. Since the toxic side effects of current L-asparaginases often result in treatment discontinuation in ALL patients, this novel ErA-TM variant with ultra-low L-glutaminase co-activity and long in vivo persistence may have great clinical potential.

L-Glutamine-, peptidyl- and protein-glutaminases: structural features and applications in the food industry.

L-Glutaminases are enzymes that catalyze the cleavage of the gamma-amido bond of L-glutamine residues, producing ammonia and L-glutamate. These enzymes have several applications in food and pharmaceutical industries. However, the L-glutaminases that hydrolyze free L-glutamine (L-glutamine glutaminases, EC 3.5.1.2) have different structures and properties with respect to the L-glutaminases that hydrolyze the same amino acid covalently bound in peptides (peptidyl glutaminases, EC 3.5.1.43) and proteins (protein-glutamine glutaminase, EC 3.5.1.44). In the food industry, L-glutamine glutaminases are applied to enhance the flavor of foods, whereas protein glutaminases are useful to improve the functional properties of proteins. This review will focus on structural backgrounds and differences between these enzymes, the methodology available to measure the activity as well as strengths and limitations. Production methods, applications, and challenges in the food industry will be also discussed. This review will provide useful information to search and identify the suitable L-glutaminase that best fits to the intended application.

Discovery of novel Glutaminase allosteric inhibitors through drug repurposing and comparative MMGB/PBSA and molecular dynamics simulation.

GLS1 enzymes (Glutaminase C (GAC) and kidney-type Glutaminase (KGA)) are gaining prominence as a target for tumor treatment including lung, breast, kidney, prostate, and colorectal. To date, several medicinal chemistry studies are being conducted to develop new and effective inhibitors against GLS1 enzymes. Telaglenastat, a drug that targets the allosteric site of GLS1, has undergone clinical trials for the first time for the therapy of solid tumors and hematological malignancies. A comprehensive computational investigation is performed to get insights into the inhibition mechanism of the Telaglenastat. Some novel inhibitors are also proposed against GLS1 enzymes using the drug repurposing approach using 2D-fingerprinting virtual screening method against 2.4 million compounds, application of pharmacokinetics, Molecular Docking, and Molecular Dynamic (MD) Simulations. A TIP3P water box of 10 Å was defined to solvate both enzymes to improve MD simulation reliability. The dynamics results were validated further by the MMGB/PBSA binding free energy method, RDF, and AFD analysis. Results of these computational analysis revealed a stable binding affinity of Telaglenastat, as well as an FDA approved drug Astemizole (IC50 ∼ 0.9 nM) and a novel para position oriented methoxy group containing Chembridge compound (Chem-64284604) that provides an effective inhibitory action against GAC and KGA.

GTP-Dependent Regulation of CTP Synthase: Evolving Insights into Allosteric Activation and NH3 Translocation.

Cytidine-5'-triphosphate (CTP) synthase (CTPS) is the class I glutamine-dependent amidotransferase (GAT) that catalyzes the last step in the de novo biosynthesis of CTP. Glutamine hydrolysis is catalyzed in the GAT domain and the liberated ammonia is transferred via an intramolecular tunnel to the synthase domain where the ATP-dependent amination of UTP occurs to form CTP. CTPS is unique among the glutamine-dependent amidotransferases, requiring an allosteric effector (GTP) to activate the GAT domain for efficient glutamine hydrolysis. Recently, the first cryo-electron microscopy structure of Drosophila CTPS was solved with bound ATP, UTP, and, notably, GTP, as well as the covalent adduct with 6-diazo-5-oxo-l-norleucine. This structural information, along with the numerous site-directed mutagenesis, kinetics, and structural studies conducted over the past 50 years, provide more detailed insights into the elaborate conformational changes that accompany GTP binding at the GAT domain and their contribution to catalysis. Interactions between GTP and the L2 loop, the L4 loop from an adjacent protomer, the L11 lid, and the L13 loop (or unique flexible "wing" region), induce conformational changes that promote the hydrolysis of glutamine at the GAT domain; however, direct experimental evidence on the specific mechanism by which these conformational changes facilitate catalysis at the GAT domain is still lacking. Significantly, the conformational changes induced by GTP binding also affect the assembly and maintenance of the NH3 tunnel. Hence, in addition to promoting glutamine hydrolysis, the allosteric effector plays an important role in coordinating the reactions catalyzed by the GAT and synthase domains of CTPS.

High-resolution structures of mitochondrial glutaminase C tetramers indicate conformational changes upon phosphate binding.

The mitochondrial enzyme glutaminase C (GAC) is upregulated in many cancer cells to catalyze the first step in glutamine metabolism, the hydrolysis of glutamine to glutamate. The dependence of cancer cells on this transformed metabolic pathway highlights GAC as a potentially important therapeutic target. GAC acquires maximal catalytic activity upon binding to anionic activators such as inorganic phosphate. To delineate the mechanism of GAC activation, we used the tryptophan substitution of tyrosine 466 in the catalytic site of the enzyme as a fluorescent reporter for glutamine binding in the presence and absence of phosphate. We show that in the absence of phosphate, glutamine binding to the Y466W GAC tetramer exhibits positive cooperativity. A high-resolution X-ray structure of tetrameric Y466W GAC bound to glutamine suggests that cooperativity in substrate binding is coupled to tyrosine 249, located at the edge of the catalytic site (i.e., the "lid"), adopting two distinct conformations. In one dimer within the GAC tetramer, the lids are open and glutamine binds weakly, whereas, in the adjoining dimer, the lids are closed over the substrates, resulting in higher affinity interactions. When crystallized in the presence of glutamine and phosphate, all four subunits of the Y466W GAC tetramer exhibited bound glutamine with closed lids. Glutamine can bind with high affinity to each subunit, which subsequently undergo simultaneous catalysis. These findings explain how the regulated transitioning of GAC between different conformational states ensures that maximal catalytic activity is reached in cancer cells only when an allosteric activator is available.

The Catalytic Mechanism of Pdx2 Glutaminase Driven by a Cys-His-Glu Triad: A Computational Study.

The catalytic mechanism of Pdx2 was studied with atomic detail employing the computational ONIOM hybrid QM/MM methodology. Pdx2 employs a Cys-His-Glu catalytic triad to deaminate glutamine to glutamate and ammonia - the source of the nitrogen of pyridoxal 5'-phosphate (PLP). This enzyme is, therefore, a rate-limiting step in the PLP biosynthetic pathway of Malaria and Tuberculosis pathogens that rely on this mechanism to obtain PLP. For this reason, Pdx2 is considered a novel and promising drug target to treat these diseases. The results obtained show that the catalytic mechanism of Pdx2 occurs in six steps that can be divided into four stages: (i) activation of Cys87 , (ii) deamination of glutamine with the formation of the glutamyl-thioester intermediate, (iii) hydrolysis of the formed intermediate, and (iv) enzymatic turnover. The kinetic data available in the literature (19.1-19.5 kcal mol-1 ) agree very well with the calculated free energy barrier of the hydrolytic step (18.2 kcal.mol-11 ), which is the rate-limiting step of the catalytic process when substrate is readily available in the active site. This catalytic mechanism differs from other known amidases in three main points: i) it requires the activation of the nucleophile Cys87 to a thiolate; ii) the hydrolysis occurs in a single step and therefore does not require the formation of a second tetrahedral reaction intermediate, as it is proposed, and iii) Glu198 does not have a direct role in the catalytic process. Together, these results can be used for the synthesis of new transition state analogue inhibitors capable of inhibiting Pdx2 and impair diseases like Malaria and Tuberculosis.

New insights into the molecular mechanisms of glutaminase C inhibitors in cancer cells using serial room temperature crystallography.

Cancer cells frequently exhibit uncoupling of the glycolytic pathway from the TCA cycle (i.e., the "Warburg effect") and as a result, often become dependent on their ability to increase glutamine catabolism. The mitochondrial enzyme Glutaminase C (GAC) helps to satisfy this 'glutamine addiction' of cancer cells by catalyzing the hydrolysis of glutamine to glutamate, which is then converted to the TCA-cycle intermediate α-ketoglutarate. This makes GAC an intriguing drug target and spurred the molecules derived from bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (the so-called BPTES class of allosteric GAC inhibitors), including CB-839, which is currently in clinical trials. However, none of the drugs targeting GAC are yet approved for cancer treatment and their mechanism of action is not well understood. Here, we shed new light on the underlying basis for the differential potencies exhibited by members of the BPTES/CB-839 family of compounds, which could not previously be explained with standard cryo-cooled X-ray crystal structures of GAC bound to CB-839 or its analogs. Using an emerging technique known as serial room temperature crystallography, we were able to observe clear differences between the binding conformations of inhibitors with significantly different potencies. We also developed a computational model to further elucidate the molecular basis of differential inhibitor potency. We then corroborated the results from our modeling efforts using recently established fluorescence assays that directly read out inhibitor binding to GAC. Together, these findings should aid in future design of more potent GAC inhibitors with better clinical outlook.

Purification and anticancer activity of glutaminase and urease free intracellular l-asparaginase from Chaetomium sp.

l-asparaginase is a chemotherapeutic drug used in the treatment of acute lymphoblastic leukemia, a malignant disorder in children. l-asparaginase helps in removing acrylamide found in fried and baked foods which is carcinogenic in nature. The search for new therapeutic enzymes is of great interest in both medical and food applications. The present work aims to isolate the intracellular l-asparaginase from endophytic fungi Chaetomium sp. The intracellular enzyme was partially purified by chromatographic techniques. Molecular weight of enzyme was found to be ~66 kDa by SDS PAGE analysis. The enzyme is highly specific for l-asparagine and did not show glutaminase and urease activity. Maximum enzyme activity was found to be 58 ± 5 U/mL at 40 °C, pH 7.0 with 2 µg of protein. Intracellular l-asparaginase from Chaetomium sp. exhibited anticancer activity on human blood cancer (MOLT-4) cells.

Circumventing the side effects of L-asparaginase.

L-asparaginase is an enzyme that catalyzes the degradation of asparagine and successfully used in the treatment of acute lymphoblastic leukemia. L-asparaginase toxicity is either related to hypersensitivity to the foreign protein or to a secondary L-glutaminase activity that causes inhibition of protein synthesis. PEGylated versions have been incorporated into the treatment protocols to reduce immunogenicity and an alternative L-asparaginase derived from Dickeya chrysanthemi is used in patients with anaphylactic reactions to the E. coli L-asparaginase. Alternative approaches commonly explore new sources of the enzyme as well as the use of protein engineering techniques to create less immunogenic, more stable variants with lower L-glutaminase activity. This article reviews the main strategies used to overcome L-asparaginase shortcomings and introduces recent tools that can be used to create therapeutic enzymes with improved features.

Structure and activation mechanism of the human liver-type glutaminase GLS2.

Cancer cells exhibit an altered metabolic phenotype, consuming higher levels of the amino acid glutamine. This metabolic reprogramming depends on increased mitochondrial glutaminase activity to convert glutamine to glutamate, an essential precursor for bioenergetic and biosynthetic processes in cells. Mammals encode the kidney-type (GLS) and liver-type (GLS2) glutaminase isozymes. GLS is overexpressed in cancer and associated with enhanced malignancy. On the other hand, GLS2 is either a tumor suppressor or an oncogene, depending on the tumor type. The GLS structure and activation mechanism are well known, while the structural determinants for GLS2 activation remain elusive. Here, we describe the structure of the human glutaminase domain of GLS2, followed by the functional characterization of the residues critical for its activity. Increasing concentrations of GLS2 lead to tetramer stabilization, a process enhanced by phosphate. In GLS2, the so-called "lid loop" is in a rigid open conformation, which may be related to its higher affinity for phosphate and lower affinity for glutamine; hence, it has lower glutaminase activity than GLS. The lower affinity of GLS2 for glutamine is also related to its less electropositive catalytic site than GLS, as indicated by a Thr225Lys substitution within the catalytic site decreasing the GLS2 glutamine concentration corresponding to half-maximal velocity (K0.5). Finally, we show that the Lys253Ala substitution (corresponding to the Lys320Ala in the GLS "activation" loop, formerly known as the "gating" loop) renders a highly active protein in stable tetrameric form. We conclude that the "activation" loop, a known target for GLS inhibition, may also be a drug target for GLS2.

Purification, characterization, and anticancer and antioxidant activities of L-glutaminase from Aspergillus versicolor Faesay4.

L-Glutaminase is an amidohydrolase which can act as a vital chemotherapeutic agent against various malignancies. In the present work, L-glutaminase productivity from Aspergillus versicolor Faesay4 was significantly increased by 7.72-fold (from 12.33 ± 0.47 to 95.15 ± 0.89 U/mL) by optimizing submerged fermentation parameters in Czapek's Dox (CZD) medium including an incubation period from 3 (12.33 ± 0.47 U/mL) to 6 days (23.36 ± 0.58 U/mL), an incubation temperature from 30 °C (23.36 ± 0.49 U/mL) to 25 °C (31.08 ± 0.60 U/mL), initial pH from pH 5.0 (8.49 ± 0.21 U/mL)  to pH 7.0 (32.18 ± 0.57 U/mL), replacement of glucose (30.19 ± 0.52 U/mL) by sucrose (48.97 ± 0.67 U/mL) as the carbon source at a concentration of 2.0% (w/v), increasing glutamine concentration as the nitrogen source from 1.0% (w/v, 48.54 ± 0.48 U/mL) to 1.5% (w/v, 63.01 ± 0.60 U/mL), and addition of a mixture of KH2PO4 and NaCl (0.5% w/v for both) to SZD as the metal supplementation (95.15 ± 0.89 U/mL). Faesay4 L-glutaminase was purified to yield total activity 13,160 ± 22.76 (U), specific activity 398.79 ± 9.81 (U/mg of protein), and purification fold 2.1 ± 3.18 with final enzyme recovery 57.22 ± 2.17%. The pure enzyme showed a molecular weight of 61.80 kDa, and it was stable and retained 100.0% of its activity at a temperature ranged from 10 to 40 °C and pH 7.0. In our trials, to increase the enzyme activity by optimizing the assay conditions (which were temperature 60 °C, pH 7.0, substrate glutamine, substrate concentration 1.0%, and reaction time 60 min), the enzyme activity increased by 358.8% after changing the assay temperature from 60 to 30 °C and then increased by 138% after decreasing the reaction time from 60 to 40 min. However, both pH 7.0 and glutamine as the substrate remain the best assay parameters for the L-glutaminase activity. When the glutamine in the assay as the reaction substrate was replaced by asparagine, lysine, proline, methionine, cysteine, glycine, valine, phenylalanine, L-alanine, aspartic acid, tyrosine, and serine, the enzyme lost 23.86%, 29.0%, 31.0%, 48.3%, 50.0%, 73.6%, 74.51%, 80.42%, 82.5%, 83.43%, 88.36%, and 89.78% of its activity with glutamine, respectively. Furthermore, Mn2+, K+, Na+, and Fe3+ were enzymatic activators that increased the L-glutaminase activity by 25.0%, 18.05%, 10.97%, and 8.0%, respectively. Faesay4 L-glutaminase was characterized as a serine protease enzyme as a result of complete inhibition by all serine protease inhibitors (PMSF, benzamidine, and TLCK). Purified L-glutaminase isolated from Aspergillus versicolor Faesay4 showed potent DPPH scavenging activities with IC50 = 50 µg/mL and anticancer activities against human liver (HepG-2), colon (HCT-116), breast (MCF-7), lung (A-549), and cervical (Hela) cancer cell lines with IC50 39.61, 12.8, 6.18, 11.48, and 7.25 µg/mL, respectively.

Molecular modeling and LC-MS-based metabolomics of a glutamine-valproic acid (Gln-VPA) derivative on HeLa cells.

Glutaminase plays an important role in carcinogenesis and cancer cell growth. This biological target is interesting against cancer cells. Therefore, in this work, in silico [docking and molecular dynamics (MD) simulations] and in vitro methods (antiproliferative and LC-MS metabolomics) were employed to assay a hybrid compound derived from glutamine and valproic acid (Gln-VPA), which was compared with 6-diazo-5-oxo-L-norleucine (DON, a glutaminase inhibitor) and VPA (contained in Gln-VPA structure). Docking results from some snapshots retrieved from MD simulations show that glutaminase recognized Gln-VPA and DON. Additionally, Gln-VPA showed antiproliferative effects in HeLa cells and inhibited glutaminase activity. Finally, the LC-MS-based metabolomics studies on HeLa cells treated with either Gln-VPA (IC60 = 8 mM) or DON (IC50 = 3.5 mM) show different metabolomics behaviors, suggesting that they modulate different biological targets of the cell death mechanism. In conclusion, Gln-VPA is capable of interfering with more than one pharmacological target of cancer, making it an interesting drug that can be used to avoid multitherapy of classic anticancer drugs.

Permeabilization and immobilization of whole-cell Pseudomonas nitroreducens SP.001 to improve its l-glutaminase performance.

BACKGROUND: L-Glutaminase is considered to be an important industrial enzyme in both the pharmaceutical and food industries, especially for producing functional glutamyl compounds, such as l-theanine. Pseudomonas nitroreducens SP.001 with intracellular l-glutaminase activity has been screened previously. In the present study, three physical permeabilization methods were used to improve l-glutaminase activity. Then, the whole-cell immobilization conditions of permeabilized cells using sodium alginate as an embedding agent were optimized to enhance the enzyme's stability and reusability. The characteristics of the immobilized cells were investigated in comparison with those of permeabilized cells. RESULTS: The results obtained showed that cell permeabilization using osmotic shock with 155 g L-1 sucrose markedly improved enzyme activity. Then, an effective procedure for immobilization of permeabilized P. nitroreducens cells was established. The optimum conditions for cell immobilization were: sodium alginate 40 g L-1 , calcium chloride 30 g L-1 , cell mass 100 g L-1 and a curing time of 3 h. After successful immobilization, characterization studies revealed that the thermostability and pH resistance of l-glutaminase from immobilized cells were enhanced compared to those from permeabilized cells. Moreover, the immobilized biocatalyst could be reused up to 10 times and retained 80% of its activity. CONCLUSION: The stability and reusability of the permeabilized cells were improved through the immobilization. These findings indicated that immobilized whole-cell l-glutaminase from P. nitroreducens SP.001 possesses more potential for various industrial biotechnological applications than free cells. © 2020 Society of Chemical Industry.