Fusarium sp. L-asparaginases: purification, characterization, and potential assessment as an antileukemic chemotherapeutic agent.

Asparaginases important role in the treatment of leukemia. It is part of chemotherapy in the treatment of leukemia in the last three decades. L-Asparaginase is isolated from Fusarium sp. isolated from soil and purified using ammonium sulfate precipitation and Sephadex G 100. Characterization of the crude enzyme revealed it is a metalloprotease inhibited by EDTA. Hg2+, Cd2+, and Pb2+ also inhibited the enzyme. Mg2+, Zn2+, and Ca2+ activated L-asparaginase. Furthermore, kinetic studies of purified enzyme were carried out. Vmax and Km were 0.031 M and 454 U/mL, respectively. The optimum temperature was 30 °C and the optimum pH was 7. Concerning substrate specificity, gelatin and casein in addition to L-asparagine were tested. The enzyme was found to be nonspecific that could hydrolyze all tested substrates at different rates. The maximum enzyme activity was recorded in the case of L-asparagine, followed by casein and gelatin, respectively. The molecular weight of L-asparaginase was 22.5 kDa. The antileukemic cytotoxicity assay of the enzyme against RAW2674 leukemic cell lines by MTT viability test was estimated. The enzyme exhibited antileukemic activity with IC50 of 50.1 UmL-1. The current work presents additional information regarding the purification and characterization of the enzyme produced by Fusarium sp. and its evaluation as a potential antileukemic chemotherapeutic agent.

Implementation of the asparaginase activity assessment technique for clinical use: experience of a Brazilian Center.

Acute lymphoid leukemia is a childhood cancer that in high-income countries has event-free survival rates of 80% and global survival rates of 90%. In Brazil these rates are under 70%. This difference may be due to the implementation of supportive care, including the assessment of asparaginase (ASNase) activity. ASNase may cause hypersensitivity reactions and silent drug inactivation. For this reason, ASNase activity monitoring is an essential tool to ensure an effective treatment. Our aim was to implement an ASNase activity measurement technique at a hospital setting. samples from children who were given Escherichia coli-derived ASNase were collected. The results of the analyses conducted in our laboratory Hospital de Clínicas de Porto Alegre were compared to those of two institutions: Centro Infantil Boldrini and University of Munster. 262 samples were assessed. The results of the first analyses were compared with those obtained at Centro Infantil Boldrini and showed an ICC of 0.954. Thirty samples were sent to the University of Munster and presented an ICC was 0.960. Our results, when compared to those of national and international centers, showed an excellent agreement. The study was able to implement an ASNase activity test to monitor the treatment.

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

BACKGROUND: L-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. METHODS: Production 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. RESULTS: The 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°Ð¡, while being 62.65 min at 60°Ð¡. 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). CONCLUSIONS: The study reveals the excellent property of this enzyme which makes it highly valuable for development of chemotherapeutic drug.

Biotechnological production and practical application of L-asparaginase enzyme.

L-asparaginase is a vital enzyme of medical importance, and renowned as a chemotherapeutic agent. The relevance of this enzyme is not only limited as an anti-cancer agent, it also possesses a wide range of medical application. The application includes the antimicrobial property, treatment of infectious diseases, autoimmune diseases, canine and feline cancer. Apart from the health care industry, its significance is also established in the food sector as a food processing agent to reduce the acrylamide concentration. L-asparaginase is known to be produced from various bacterial, fungal and plant sources. However, there is a huge market demand due to its wide range of application. Therefore, the industry is still in the search of better-producing source in terms of high yield and low immunogenicity. It can be produced by both submerged and solid state fermentation, and each fermentation process has its own merits and demerits. This review paper focuses on its improved production strategy by adopting statistical experimental optimization techniques, development of recombinant strains, through mutagenesis and nanoparticle immobilization, adopting advanced and cost-effective purification techniques. Available research literature proves the competence and therapeutic potential of this enzyme. Therefore, research orientation toward the exploration of this clinical significant enzyme has to be accelerated. The objectives of this review are to discuss the high yielding sources, current production strategies, improvement of production, effective downstream processing and therapeutic application of L-asparaginase.

Current applications and different approaches for microbial l-asparaginase production.

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.

Nanostructure L-asparaginase-fatty acid bioconjugate: synthesis, preformulation study and biological assessment.

The present study aims to develop a novel L-asparaginase fatty acid bioconjugates and characterize their applicability for intravenous delivery of L-asparaginase. These bioconjugates were achieved by covalent linkage of fatty acids having different chain lengths (C12, C16 and C22) to the native enzyme. To determine the optimum conditions of bioconjugation, the effect of lipid:protein ratios, reaction time and medium composition on enzyme activity and conjugation degree were evaluated. The native and bioconjugates have been characterized by activity, conjugation degree, particle size, and zeta potential. The results showed that bioconjugated L-asparaginase were more resistant to proteolysis, more stable at different pH, and had prolonged plasma half-life, compared to the native form. From partition coefficient study, the modified enzymes showed approximately 15-fold increase in hydrophobicity. Secondary structure analysis using circular dichroism revealed alteration after lipid conjugation. In addition, the Michaelis constant of the native enzyme was 3.38 mM, while the bioconjugates showed the higher affinity to the substrate L-asparagine. These findings indicate that new lipid bioconjugation could be a very useful strategy for intravenous delivery of L-asparaginase.

Hidden Markov models for evolution and comparative genomics analysis.

The problem of reconstruction of ancestral states given a phylogeny and data from extant species arises in a wide range of biological studies. The continuous-time Markov model for the discrete states evolution is generally used for the reconstruction of ancestral states. We modify this model to account for a case when the states of the extant species are uncertain. This situation appears, for example, if the states for extant species are predicted by some program and thus are known only with some level of reliability; it is common for bioinformatics field. The main idea is formulation of the problem as a hidden Markov model on a tree (tree HMM, tHMM), where the basic continuous-time Markov model is expanded with the introduction of emission probabilities of observed data (e.g. prediction scores) for each underlying discrete state. Our tHMM decoding algorithm allows us to predict states at the ancestral nodes as well as to refine states at the leaves on the basis of quantitative comparative genomics. The test on the simulated data shows that the tHMM approach applied to the continuous variable reflecting the probabilities of the states (i.e. prediction score) appears to be more accurate then the reconstruction from the discrete states assignment defined by the best score threshold. We provide examples of applying our model to the evolutionary analysis of N-terminal signal peptides and transcription factor binding sites in bacteria. The program is freely available at http://bioinf.fbb.msu.ru/~nadya/tHMM and via web-service at http://bioinf.fbb.msu.ru/treehmmweb.