Studies on the recombinant production and anticancer activity of thermostable L- asparaginase I from Pyrococcus abyssi / Estudos sobre a produção recombinante e atividade anticancerígena da L-asparaginase I termoestável de Pyrococcus abyssi

L-Asparaginase catalysing the breakdown of L-Asparagine to L-Aspartate and ammonia is an enzyme of therapeutic importance in the treatment of cancer, especially the lymphomas and leukaemia. The present study describes the recombinant production, properties and anticancer potential of enzyme from a hyperthermophilic archaeon Pyrococcus abyssi. There are two genes coding for asparaginase in the genome of this organism. A 918 bp gene encoding 305 amino acids was PCR amplified and cloned in BL21 (DE3) strain of E. coli using pET28a (+) plasmid. The production of recombinant enzyme was induced under 0.5mM IPTG, purified by selective heat denaturation and ion exchange chromatography. Purified enzyme was analyzed for kinetics, in silico structure and anticancer properties. The recombinant enzyme has shown a molecular weight of 33 kDa, specific activity of 1175 U/mg, KM value 2.05mM, optimum temperature and pH 80°C and 8 respectively. No detectable enzyme activity found when L-Glutamine was used as the substrate. In silico studies have shown that the enzyme exists as a homodimer having Arg11, Ala87, Thr110, His112, Gln142, Leu172, and Lys232 being the putative active site residues. The free energy change calculated by molecular docking studies of enzyme and substrate was found as ∆G ­ 4.5 kJ/mole indicating the affinity of enzyme with the substrate. IC50 values of 5U/mL to 7.5U/mL were determined for FB, caco2 cells and HepG2 cells. A calculated amount of enzyme (5U/mL) exhibited 78% to 55% growth inhibition of caco2 and HepG2 cells. In conclusion, the recombinant enzyme produced and characterized in the present study offers a good candidate for the treatment of cancer. The procedures adopted in the present study can be prolonged for in vivo studies.

A L-asparaginase, que catalisa a degradação da L-asparagina em L-aspartato e amônia, é uma enzima de importância terapêutica no tratamento do câncer, especialmente dos linfomas e da leucemia. O presente estudo descreve a produção recombinante, propriedades e potencial anticancerígeno da enzima de Pyrococcus abyssi, um archaeon hipertermofílico. Existem dois genes que codificam para a asparaginase no genoma desse organismo. Um gene de 918 bp, que codifica 305 aminoácidos, foi amplificado por PCR e clonado na cepa BL21 (DE3) de E. coli usando o plasmídeo pET28a (+). A produção da enzima recombinante foi induzida sob 0,5mM de IPTG, purificada por desnaturação seletiva por calor e cromatografia de troca iônica. A enzima purificada foi analisada quanto à cinética, estrutura in silico e propriedades anticancerígenas. A enzima recombinante apresentou peso molecular de 33 kDa, atividade específica de 1.175 U / mg, valor de KM 2,05 mM, temperatura ótima de 80º C e pH 8. Nenhuma atividade enzimática detectável foi encontrada quando a L-glutamina foi usada como substrato. Estudos in silico mostraram que a enzima existe como um homodímero, com Arg11, Ala87, Thr110, His112, Gln142, Leu172 e Lys232 sendo os resíduos do local ativo putativo. A mudança de energia livre calculada por estudos de docking molecular da enzima e do substrato foi encontrada como ∆G ­ 4,5 kJ / mol, indicando a afinidade da enzima com o substrato. Valores de IC50 de 5U / mL a 7,5U / mL foram determinados para células FB, células caco2 e células HepG2. Uma quantidade de enzima (5U / mL) apresentou inibição de crescimento de 78% a 55% das células caco2 e HepG2, respectivamente. Em conclusão, a enzima recombinante produzida e caracterizada no presente estudo é uma boa possibilidade para o tratamento do câncer. Os procedimentos adotados na presente pesquisa podem ser aplicados para estudos in vivo.

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

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.

Current applications and different approaches for microbial L-asparaginase production

ABSTRACT L-asparaginase (EC 3.5.1.1) is an enzyme that catalysis mainly the asparagine hydrolysis in L-aspartic acid and ammonium. This enzyme is presented in different organisms, such as microorganisms, vegetal, and some animals, including certain rodent's serum, but not unveiled in humans. It can be used as important chemotherapeutic agent for the treatment of a variety of lymphoproliferative disorders and lymphomas (particularly acute lymphoblastic leukemia (ALL) and Hodgkin's lymphoma), and has been a pivotal agent in chemotherapy protocols from around 30 years. Also, other important application is in food industry, by using the properties of this enzyme to reduce acrylamide levels in commercial fried foods, maintaining their characteristics (color, flavor, texture, security, etc.) Actually, L-asparaginase catalyzes the hydrolysis of L-asparagine, not allowing the reaction of reducing sugars with this aminoacid for the generation of acrylamide. Currently, production of L-asparaginase is mainly based in biotechnological production by using some bacteria. However, industrial production also needs research work aiming to obtain better production yields, as well as novel process by applying different microorganisms to increase the range of applications of the produced enzyme. Within this context, this mini-review presents L-asparaginase applications, production by different microorganisms and some limitations, current investigations, as well as some challenges to be achieved for profitable industrial production.

Statistical and evolutionary optimization for enhanced production of an anti-leukemic enzyme, L-asparaginase, in a protease-deficient Bacillus aryabhattai ITBHU02 isolated from the soil contaminated with hospital waste.

Over the past few decades, L-asparaginase has emerged as an excellent anti-neoplastic agent. In present study, a new strain ITBHU02, isolated from soil site near degrading hospital waste, was investigated for the production of extracellular L-asparaginase. Further, it was renamed as Bacillus aryabhattai ITBHU02 based on its phenotypical features, biochemical characteristics, fatty acid methyl ester (FAME) profile and phylogenetic similarity of 16S rDNA sequences. The strain was found protease-deficient and its optimal growth occurred at 37 °C and pH 7.5. The strain was capable of producing enzyme L-asparaginase with maximum specific activity of 3.02±0.3 Umg-1 protein, when grown in un-optimized medium composition and physical parameters. In order to improve the production of L-asparaginase by the isolate, response surface methodology (RSM) and genetic algorithm (GA) based techniques were implemented. The data achieved through the statistical design matrix were used for regression analysis and analysis of variance studies. Furthermore, GA was implemented utilizing polynomial regression equation as a fitness function. Maximum average L-asparaginase productivity of 6.35 Umg-1 was found at GA optimized concentrations of 4.07, 0.82, 4.91, and 5.2 gL‑1 for KH2PO4, MgSO4.7H2O, L-asparagine, and glucose respectively. The GA optimized yield of the enzyme was 7.8% higher in comparison to the yield obtained through RSM based optimization.

Studies on the asparaginolytic enzymes of streptomycetes. I-Culture conditions for the production of L-asparaginase enzyme from streptomyces longsporusflavus [F-15] strain

Asparaginolytic activity of seven streptomyces strains was studied under static culture condition on glycerol-L-asparagine [GA] medium. Streptomyces longsporusflavus [F-15] only showed the highest asparagenolytic activity. Data revealed that the maximal yield of L-aspara- ginase from this strain can be obtained by growing it on starch-L-asparagine-yeast extract [SAY] medium, containing [W/V]: 1.5% starch, 0.2% L-asparagine, 0.05% yeast extract, 0.1% K2HP04.3H2O, 0.0001% FeSO4.7H2.0,0.0001% MnC12.4H2O,0001% ZnSO4 71120 which was initially adjusted to pH 7.0, inoculated by 2% [VM of homogenized spore suspension [containing approximately 2.5 x 10[6] spores/ml] of 4 days old culture on starch nitrate medium and incubated at 30§ for 5 days, under static culture condition

Fermentative production and isolation of L-asparaginase from Erwinia carotovora, EC-113.

L-Asparaginase, an enzyme-drug used for the treatment of acute lymphoblastic leukemia was isolated from Erwinia carotovora. The effects of different carbon and nitrogen sources on the fermentative production of the enzyme were studied. Lactose, monosodium glutamate, corn steep liquor, tryptone and yeast extract showed significant stimulation of the production. When L-asparagine (0.2%), a substrate of the enzyme was added to a fermentation medium, a mutant strain EC-113 exhibited 6 times higher production indicating a distinct induction. The enzyme was extracted from the cells and purified about 30 fold to apparent homogeneity employing polyacrylamide gel electrophoresis. The methods used in sequence were DEAE cellulose chromatography, sephadex G-200 gel filtration, hydroxylapatite ion-exchange and affinity chromatography on sepharose CL-6B. The recovery of enzyme was 60%. The purified enzyme showed optimal pH at 8.0 and optimal temperature at 50 degrees C. The Km value of purified enzyme was 1.8 x 10(-5) M. LD50 of purified enzyme in mice by intravenous route was 4,80,000 IU/Kg and repeated treatment at 20,000 IU/Kg by intravenous route did not elicit bone marrow depression or damage to intestinal mucosa. The plasma half life was 14-24 hours and clearance time was 4-5 hours. Purified enzyme shows significant antitumor activity on experimental animal models.

Induction of L-asparaginase synthesis in Vibrio proteus.

Studies on L-asparaginase synthesis in V. proteus showed increased synthesis in cultures grown under conditions of moderate aeration (P less than 0.005) after oxygen had been used up from the medium. Addition of sodium lactate to the medium at a concentration of 80 mu mole/ml, stimulated L-asparaginase synthesis (2.2 times over control) in moderately-aerated cultures (P less than 0.001). The substrate L-asparagine induced enzyme synthesis when growth conditions were made anaerobic or lactate was incorporated into the medium (3.8 times increased enzyme synthesis over control).