Glutaminase Free L-asparaginase Research Articles

Enhanced production of glutaminase-free l-asparaginase by marine Bacillus velezensis and cytotoxic activity against breast cancer cell lines

Abstract Background The increasing rate of breast cancer globally requires extraordinary efforts to discover new effective sources of chemotherapy with fewer side effects. Glutaminase-free l -asparaginase is a vital chemotherapeutic agent for various tumor malignancies. Microorganisms from extreme sources, such as marine bacteria, might have high l -asparaginase productivity and efficiency with exceptional antitumor action toward breast cancer cell lines. Results l -Asparaginase-producing bacteria, Bacillus velezensis isolated from marine sediments, were identified by 16S rRNA sequencing. l -Asparaginase production by immobilized cells was 61.04% higher than that by free cells fermentation. The significant productivity of enzyme occurred at 72 h, pH 6.5, 37°C, 100 rpm. Optimum carbon and nitrogen sources for enzyme production were glucose and NH4Cl, respectively. l -Asparaginase was free from glutaminase activity, which was crucial medically in terms of their severe side effects. The molecular weight of the purified enzyme is 39.7 KDa by SDS-PAGE analysis and was ideally active at pH 7.5 and 37°C. Notwithstanding, the highest stability of the enzyme was found at pH 8.5 and 70°C for 1 h. The enzyme kinetic parameters displayed Vmax at 41.49 μmol/mL/min and a Km of 3.6 × 10−5 M, which serve as a proof of the affinity to its substrate. The anticancer activity of the enzyme against breast adenocarcinoma cell lines demonstrated significant activity toward MDA-MB-231 cells when compared with MCF-7 cells with IC50 values of 12.6 ± 1.2 μg/mL and 17.3 ± 2.8 μg/mL, respectively. Conclusion This study provides the first potential of glutaminase-free l -asparaginase production from the marine bacterium Bacillus velezensis as a prospect anticancer pharmaceutical agent for two different breast cancer cell lines. How to cite: Mostafa Y, Alrumman S, Alamri S, et al. Enhanced production of glutaminase-free L-asparaginase by marine Bacillus velezensis and cytotoxic activity against breast cancer cell lines. Electron J Biotechnol 2019;42. https://doi.org/10.1016/j.ejbt.2019.10.001 .

Cationic reverse micellar based purification of recombinant glutaminase free L-asparaginase II of Bacillus subtilis WB800N from fermentation media

Reverse micellar extraction (RME), a liquid-liquid based separation is a versatile tool for protein purification. A statistical approach was employed for the purification of recombinant glutaminase free anti-cancerous enzyme viz., l-asparaginase II to evaluate the effects of RME in current study. The cationic system (CTAB/iso-octane/hexanol/butanol) was used in RME to optimize both forward and backward protein extraction efficiency. By adapting Taguchi's orthogonal array (OA), maximum forward extraction efficiency (FEE) of 86.98% with 84.82% enzyme activity recovery and 1.04 times purification fold achieved with the optimized parameters. Under the optimal levels, the back extraction efficiency (BEE) was observed to be 96.97% with 93.07% enzyme activity recovery and 1.38 times purification fold. Further, mass transfer kinetic studies of RME indicated the mass transfer coefficients of forward and backward extraction to be 0.049 min−1 and 0.036 min−1 respectively.

Purification, characterization and immunogenicity assessment of glutaminase free L-asparaginase from Streptomyces brollosae NEAE-115

BackgroundL-asparaginase is a potential therapeutic enzyme widely used in the chemotherapy protocols of pediatric and adult patients with acute lymphoblastic leukemia. However, its use has been limited by a high rate of hypersensitivity in the long-term used. Hence, there is a continuing need to search for other L-asparaginase sources capable of producing an enzyme with less adverse effects.MethodsProduction of extracellular L-asparaginase by Streptomyces brollosae NEAE-115 was carried out using submerged fermentation. L-asparaginase was purified by ammonium sulphate precipitation and pure enzyme was reached using ion-exchange chromatography, followed by enzyme characterization. Anticancer activity towards Ehrlich Ascites Carcinoma (EAC) cells was investigated in female Swiss albino mice by determination of tumor size and the degree of tumor growth inhibition. The levels of anti-L-asparaginase IgG antibodies in mice sera were measured using ELISA method.ResultsThe purified L-asparaginase showed a total activity of 795.152 with specific activity of 76.671 U/mg protein and 7.835 − purification fold. The enzyme purity was confirmed by using SDS–PAGE separation which revealed only one distinctive band with a molecular weight of 67 KDa. The enzyme showed maximum activity at pH 8.5, optimum temperature of 37 °C, incubation time of 50 min and optimum substrate concentration of 7 mM. A Michaelis-Menten constant analysis showed a Km value of 2.139 × 10− 3 M with L-asparagine as substrate and Vmax of 152.6 UmL− 1 min− 1. The half-life time (T1/2) was 65.02 min at 50°С, while being 62.65 min at 60°С. Furthermore, mice treated with Streptomyces brollosae NEAE-115 L-asparaginase showed higher cytotoxic effect (79% tumor growth inhibition) when compared to commercial L-asparaginase group (67% tumor growth inhibition).ConclusionsThe study reveals the excellent property of this enzyme which makes it highly valuable for development of chemotherapeutic drug.

Glutaminase free l-asparaginase from Vibrio cholerae: Heterologous expression, purification and biochemical characterization

l-asparaginase is used as a pivotal agent in treatment of lympho proliferative disorders and holds an excessive demand for food processing aids. Most of the l-asparaginases possess glutaminase activity which might lead to side effects during the treatment. The search for new therapeutic enzymes is of great interest in both medical and food applications. In this study, Vibrio cholerael-asparaginase was recombinantly overexpressed in E. coli, purified to homogeneity and molecular size was estimated to be 36.6kDa. Recombinant enzyme showed an optimum pH and temperature of 7.0 and 37°C respectively. The Km and Vmax of the enzyme for l-asparagine was 1.1mM and 1006μM/min respectively. The enzyme is highly specific for l-asparagine and did not show glutaminase activity. The enzyme possessed high half life time and good stability over a wide range of physiological conditions. Tm of the enzyme was found to be 81°C by circular dichroism and differential scanning calorimetric techniques. This enzyme possesses novel properties such as (i) glutaminase free l-asparaginase activity, (ii) very high Vmax value with high affinity and (iii) high stability at 30°C (half-life time of 2.9days).

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Enhanced production of glutaminase free L-asparaginase II by Bacillus subtilis WB800N through media optimization

We studied the crucial components which elevate the expression of recombinant novel glutaminase free L-asparaginase II (rL-asp II) from Bacillus subtilis WB800N. The Plackett-Burman tool identified sucrose, NH4Cl, NaH2-PO4 and MgSO4 as the significant influencing factors (p<0.05). Further investigations showed that artificial neural network-genetic algorithm (ANN-GA) was more effective than central composite design (CCD) in optimizing the influencing factors. The maximum rL-asp II expression was found to be 389.56 IU/ml and 525.98 IU/ml using CCD (R2=90.4%) and ANN-GA (R2=96.2%), respectively. The validation experiments were carried out in a 3 L batch bioreactor where kinetic modelling of the obtained data was done. The rL-asp II expressed effectively inhibiting the polyacrylamide formation in vitro where no solidification was observed, when 2ml of purified rL-asp II used even after 60 min of incubation. This is the first study to report highest production of rL-asp II in B. subtilis WB800N (525.98 IU/ml) till date by combining statistical designs with consecutive intermittent addition of IPTG in batch reactor.

Batch and fed-batch bioreactor studies for the enhanced production of glutaminase-free L-asparaginase from Pectobacterium carotovorum MTCC 1428

ABSTRACTThe effect of dissolved oxygen (DO) level and pH (controlled/uncontrolled) was first studied to enhance the production of novel glutaminase-free L-asparaginase by Pectobacterium carotovorum MTCC 1428 in a batch bioreactor. The optimum level of DO was found to be 20%. The production of L-asparaginase was found to be maximum when pH of the medium was maintained at 8.5 after 12 h of fermentation. Under these conditions, P. carotovorum produced 17.97 U/mL of L-asparaginase corresponding to the productivity of 1497.50 U/L/h. The production of L-asparaginase was studied in fed-batch bioreactor by feeding L-asparagine (essential substrate for production) and/or glucose (carbon source for growth) at the end of the reaction period of 12 h. The initial medium containing both L-asparagine and glucose in the batch mode and L-asparagine in the feeding stream was found to be the best combination for enhanced production of glutaminase-free L-asparaginase. Under this condition, the L-asparaginase production was increased to 38.8 U/mL, which corresponded to a productivity of 1615.8 U/L/h. The production and productivity were increased by 115.8% and 7.9%, respectively, both of which are higher than those obtained in the batch bioreactor experiments.

Kinetics of growth on dual substrates, production of novel glutaminase-free L-asparaginase and substrates utilization by Pectobacterium carotovorum MTCC 1428 in a batch bioreactor

Bacterial L-asparaginase has been widely used as a potential therapeutic agent in the treatment of various lymphoblastic leukemia diseases. We studied product and dual substrates utilization kinetics by P. carotovorum MTCC 1428 in batch bioreactor. The kinetic study revealed that the maximum growth of P. carotovorum MTCC 1428 was achieved at 2 g l−1 and 5 g l−1 of glucose and L-asparagine, respectively. Different substrate inhibition models were fitted to the growth kinetic data and the additive form of double Luong model was found to best explain the growth kinetics of P. carotovorum MTCC 1428. The kinetic parameters of growth studies showed that the predicted maximum inhibition concentration of glucose (Smg) and L-asparagine (Sma) was close to the experimentally observed value 15.0 and 10 g l−1, respectively. Modified form of the Luedeking-Piret model was used to describe the kinetics of L-asparaginase production, and the system seems to be mixed growth associated. Kinetic models of dual substrate growth, L-asparaginase production and substrate(s) utilization by P. carotovorum MTCC 1428 well fitted with experimental data with regression coefficients (R2) value of 0.97, 0.96 and 0.93, respectively.

Hemocompatible glutaminase freel-asparaginase from marine Bacillus tequilensis PV9W with anticancer potential modulating p53 expression

Glutaminase freel-asparaginase from a marine isolateBacillus tequilensisPV9W: production, purification, characterization and its biological applications.

Correction: Hemocompatible glutaminase free l-asparaginase from marine Bacillus tequilensis PV9W with anticancer potential modulating p53 expression

Correction for ‘Hemocompatible glutaminase free l-asparaginase from marine Bacillus tequilensis PV9W with anticancer potential modulating p53 expression’ by Ganeshan Shakambari et al., RSC Adv., 2016, 6, 25943–25951.

High yield expression of novel glutaminase free L-asparaginase II of Pectobacterium carotovorum MTCC 1428 in Bacillus subtilis WB800N.

Gene encoding glutaminase-free L-asparaginase II (ans B2) from Pectobacterium carotovorum MTCC 1428 was cloned into pHT43, transformed in Bacillus subtilis WB800N and optimised the expression levels of recombinant enzyme. A three-fold higher enzyme production was observed with an efficient transformant as compared to native strain. Enzyme localization studies revealed that >90% of recombinant enzyme is secreted extracellularly, a little fraction is attached to the membrane (>6%) and localised intracellularly (3%). The expression of recombinant L-asparaginase II was confirmed by SDS-PAGE, IMAC (Immobilised metal ion affinity chromatography) purification followed by Western blotting. Process parameter optimization with OFAT (one factor at a time) revealed that rpm (120), temperature (37 °C), Isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration (1 mM) and time of induction (0.8 OD600nm) plays a vital role where a maximum of 55 IU/ml was achieved. Further, consecutive induction by IPTG improved the enzyme production up to 105 IU/ml with a specific activity of 101 IU/mg of protein. Molecular modelling analysis depicted that amino acids, GLY60, GLY119 and ALA252 in the active site are responsible for the glutaminase free L-asparaginase II activity. This is the first report on enhanced expression of recombinant glutaminase-free L-asparaginase II by intermediate addition of IPTG.

Optimization of Culture Conditions for Production of the Anti-Leukemic Glutaminase Free L-Asparaginase by Newly Isolated Streptomyces olivaceus NEAE-119 Using Response Surface Methodology.

Among the antitumor drugs, bacterial enzyme L-asparaginase has been employed as the most effective chemotherapeutic agent in pediatric oncotherapy especially for acute lymphoblastic leukemia. Glutaminase free L-asparaginase producing actinomycetes were isolated from soil samples collected from Egypt. Among them, a potential culture, strain NEAE-119, was selected and identified on the basis of morphological, cultural, physiological, and biochemical properties together with 16S rRNA sequence as Streptomyces olivaceus NEAE-119 and sequencing product (1509 bp) was deposited in the GenBank database under accession number KJ200342. The optimization of different process parameters for L-asparaginase production by Streptomyces olivaceus NEAE-119 using Plackett-Burman experimental design and response surface methodology was carried out. Fifteen variables (temperature, pH, incubation time, inoculum size, inoculum age, agitation speed, dextrose, starch, L-asparagine, KNO3, yeast extract, K2HPO4, MgSO4·7H2O, NaCl, and FeSO4·7H2O) were screened using Plackett-Burman experimental design. The most positive significant independent variables affecting enzyme production (temperature, inoculum age, and agitation speed) were further optimized by the face-centered central composite design-response surface methodology.

Industrial effluent as a substrate for glutaminase freel-asparaginase production from Pseudomonas plecoglossicida strain RS1; media optimization, enzyme purification and its characterization

Glutaminase freel-asparaginase production byPseudomonas plecoglossicidaRS1 using industrial effluent as a substrate: media optimization, enzyme purification and its characterization.

Novel glutaminase free L-asparaginase from Nocardiopsis alba NIOT-VKMA08: production, optimization, functional and molecular characterization.

Studies were carried out for the optimization and production of novel extracellular glutaminase-free L-asparaginase from Nocardiopsis alba NIOT-VKMA08. Among the tested carbon and nitrogen sources, maximum L-asparaginase production was observed with a combination of L-asparagine and maltose (1.5%) and twofold increase in yield (18.47 IU mL(-1)) was observed with newly optimized NIOT-asparaginase medium. Activity of the purified enzyme was moderately inhibited by various divalent cations and thiol group blocking reagents, with K(m) and V(max) of 0.127 mM and 5.50 U µg(-1). Optimum pH and temperature of purified L-asparaginase for the hydrolysis of L-asparagine was 8.0 and 37 °C, respectively. The enzyme inhibited polyacrylamide formation in 10% solution and it was very specific for its natural substrate L-asparagine. Partial glutaminase activity was not detected, which could reduce the possibility of side effects during cancer therapy. L-Asparaginase biosynthesis gene (ansA) was cloned and transformed in E. coli JM109. The ansA gene sequence reported in this study contains several base substitutions with that of reported sequences in GenBank, resulting in altered amino acid sequences of the translated protein.

Characterization of a recombinant glutaminase-free L-asparaginase (ansA3) enzyme with high catalytic activity from Bacillus licheniformis.

L-Asparaginase (3.5.1.1) is an enzyme widely used to treat the acute lymphoblastic leukemia. Two genes coding for L-asparaginase (ansA1 and ansA3) from Bacillus licheniformis MTCC 429 were cloned and overexpressed in Escherichia coli BL21 (DE3) cells. The recombinant proteins were purified to homogeneity by one-step purification process and further characterized for various biochemical parameters. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that both the enzymes are monomers of ∼37 kDa. Recombinant ansA1 was found to be highly unstable, and recombinant ansA3 was catalytically active and stable, which showed an optimum activity of 407.65 IU/mg at 37 °C and pH 8. Recombinant ansA3 showed higher substrate specificity for L-asparagine with negligible glutaminase activity. Kinetic parameters like K m , V max, k cat, and k cat/K m were calculated for recombinant ansA3.

Screening and Isolation of Novel Glutaminase Free L-asparaginase from Fungal Endophytes

Screening and Isolation of Novel Glutaminase Free L-asparaginase from Fungal Endophytes

Studies on pH and thermal stability of novel purified L-asparaginase from Pectobacterium carotovorum MTCC 1428

Glutaminase free L-asparaginase is known to be an excellent anticancer agent. In the present study, the combined effect of pH and temperature on the performance of purified novel L-asparaginase from Pectobacterium carotovorum MTCC 1428 was studied under assay conditions using response surface methodology (RSM). Deactivation studies and thermodynamic parameters of this therapeutically important enzyme were also investigated. The optimum pH and temperature of the purified L-asparaginase were found to be 8.49 and 39.3 degrees C, respectively. The minimum deactivation rate constant (k(d)) and maximum half life (t1/2) were found to be 0.041 min(-1) and 16.9 h, respectively at pH of 8.6 and 40 degreesC. Thermodynamic parameters (deltaG, deltaH, deltaS, and activation energies) were also evaluated for purified L-asparaginase. The probable mechanism of deactivation of purified L-asparaginase was explained to an extent on the basis of deactivation studies and thermodynamic parameters.

Isolation and Purification of High Efficiency L-asparaginase by Quantitative Preparative Continuous-elution SDS PAGE Electrophoresis

An Unique extracelluar glutaminase free L-asparaginase from novel marine Actinomycetes was isolated to perceptible homogeneity in agro industrial wastes. Quantitative Preparative Continuous-Elution SDS PAGE Electrophoresis is a high-resolution method for the preparative isolation of L-Asparaginase in biological samples. The enzyme was purified 248.68-fold and showed a final specific activity of 5035.28 IU/mg with an 80.71% yield. The homotetramer enzyme has a molecular mass of 133.25 kDa and an isoelectric point of approximately 5.4.Kinetic parameters, Km and Vmax of purified L-asparaginase from Streptomyces radiopugnans MS1 were found to be 0.0598, 3.5478 IU μg - 1 respectively. The de novo sequencing strategy presented here provides a rapid and reliable means to identify proteins in Streptomyces radiopugnans MS1. The purified L-asparaginase has no glutaminase activity, which can diminish the leeway of side effects during the itinerary of anti-malignancy therapy.

Purification and characterization of glutaminase-free l-asparaginase from Pectobacterium carotovorum MTCC 1428

An intracellular glutaminase-free L-asparaginase from Pectobacterium carotovorum MTCC 1428 was isolated to apparent homogeneity. The homotetramer enzyme has a molecular mass of 144.4 kDa (MALDI-TOF MS) and an isoelectric point of approximately 8.4. The enzyme is very specific for its natural substrate, L-asparagine. The activity of L-asparaginase is activated by mono cations and various effectors including Na+, K+, L-cystine, L-histidine, glutathione and 2-mercaptoethanol whereas it is moderately inhibited by various divalent cations and thiol group blocking reagents. Kinetic parameters, Km, Vmax and kcat of purified L-asparaginase from P. carotovorum MTCC 1428 were found to be 0.657 mM, 4.45 U μg(-1) and 2.751×10(3) s(-1), respectively. Optimum pH of purified L-asparaginase for the hydrolysis of L-asparagine was in the range of 8.0-10.0, and its optimum temperature was found to be 40 °C. The purified L-asparaginase has no partial glutaminase activity, which can reduce the possibility of side effects during the course of anti-cancer therapy.

Assessment of Physical Process Conditions for Enhanced Production of Novel Glutaminase-Free L-Asparaginase from Pectobacterium carotovorum MTCC 1428

Statistically based experimental design was applied to maximize the production of glutaminase-free L-asparaginase from Pectobacterium carotovorum MTCC 1428. The effect of physical process parameters (initial pH of the medium, temperature, rpm of the shaking incubator, and inoculum size) on the production of L-asparaginase from P. carotovorum MTCC 1428 was studied using central composite design technique. The individual optimum levels of initial pH of the medium, temperature, rpm of shaking incubator, and inoculum size were found to be 6.90, 29.8°C, 157rpm, and 2.61% (v/v), respectively, for the production of L-asparaginase. After physical process parameters optimization, the production and productivity of L-asparaginase was enhanced by 26.39% (specific activity) and 10.19%, respectively. Maximization of L-asparaginase production was achieved at 12h under optimal levels of physical process parameters in shake flask level.