A new extracellular glutaminase and urease-free L-asparaginase from Meyerozyma guilliermondii.

L-Asparaginase (L-ASNase) is a potent chemotherapeutic drug employed to treat leukemia and lymphoma. Currently, L-ASNases for therapeutic use are obtained from Escherichia coli and Dickeya chrysanthemi (Erwinia chrysanthemi). Despite their therapeutic potential, enzymes from bacteria are subject to inducing immune responses, resulting in a higher number of side effects. Eukaryote producers, such as fungi, may provide therapeutic alternatives through enzymes that induce relatively less toxicity and immune responses. Additional expected benefits from yeast-derived enzymes include higher activity and stability in physiological conditions. This work describes the new potential therapeutic candidate L-ASNase from the yeast Meyerozyma guilliermondii. A statistical approach (full factorial central composite design) was used to optimize L-ASNase production, considering L-asparagine and glucose concentration, pH of the medium, and cultivation time as independent factors. In addition, the crude enzymes were biochemically characterized, in terms of temperature and optimal pH, thermostability, pH stability, and associated glutaminase or urease activities. Our results showed that enzyme production increased after supplementing a pH 4.0 medium with 1.0% L-asparagine and 0.5% glucose during 75 h of cultivation. Under these optimized conditions, L-ASNase production reached 26.01 U mL-1, which is suitable for scale-up studies. The produced L-ASNase exhibits maximal activity at 37 °C and pH 7.0 and is highly stable under physiological conditions. In addition, M. guilliermondii L-ASNase has no associated glutaminase or urease activities, demonstrating its potential as a promising antineoplastic agent.

Semi-Solid Pharmaceutical Formulations for the Delivery of Papain Nanoparticles.

Papain is a therapeutic enzyme with restricted applications due to associated allergenic reactions. Papain nanoparticles have shown to be safe for biomedical use, although a method for proper drug loading and release remains to be developed. Thus, the objective of this work was to develop and assess the stability of papain nanoparticles in a prototype semi-solid formulation suitable for dermatological or topical administrations. Papain nanoparticles of 7.0 ± 0.1 nm were synthesized and loaded into carboxymethylcellulose- and poly(vinyl alcohol)-based gels. The formulations were then assayed for preliminary stability, enzyme activity, cytotoxicity studies, and characterized according to their microstructures and protein distribution. The formulations were suitable for papain nanoparticle loading and provided a stable environment for the nanoparticles. The enzyme distribution along the gel matrix was homogeneous for all the formulations, and the proteolytic activity was preserved after the gel preparation. Both gels presented a slow release of the papain nanoparticles for four days. Cell viability assays revealed no potential cytotoxicity, and the presence of the nanoparticles did not alter the microstructure of the gel. The developed systems presented a potential for biomedical applications, either as drug delivery systems for papain nanoparticles and/or its complexes.

The Place for Enzymes and Biologically Active Peptides from Marine Organisms for Application in Industrial and Pharmaceutical Biotechnology.

Since the beginning of written history, diverse texts have reported the use of enzymatic preparations in food processing and have described the medicinal properties of crude and fractionated venoms to treat various diseases and injuries. With the biochemical characterization of enzymes from distinct sources and bioactive polypeptides from animal venoms, the last sixty years have testified the advent of industrial enzymology and protein therapeutics, which are currently applicable in a wide variety of industrial processes, household products, and pharmaceuticals. Bioprospecting of novel biocatalysts and bioactive peptides is propelled by their unsurpassed properties that are applicable for current and future green industrial processes, biotechnology, and biomedicine. The demand for both novel enzymes with desired characteristics and novel peptides that lead to drug development, has experienced a steady increase in response to the expanding global market for industrial enzymes and peptidebased drugs. Moreover, although largely unexplored, oceans and marine realms, with their unique ecosystems inhabited by a large variety of species, including a considerable number of venomous animals, are recognized as untapped reservoirs of molecules and macromolecules (enzymes and bioactive venom-derived peptides) that can potentially be converted into highly valuable biopharmaceutical products. In this review, we have focused on enzymes and animal venom (poly)peptides that are presently in biotechnological use, and considering the state of prospection of marine resources, on the discovery of useful industrial biocatalysts and drug leads with novel structures exhibiting selectivity and improved performance.

Fungal production of the anti-leukemic enzyme L-asparaginase: from screening to medium development / Produção fúngica da enzima antileucêmica L-asparaginase: da triagem ao desenvolvimento de um meio de cultivo

The treatment of acute lymphoblastic leukemia is challenging due to side effects, efficacy of the available drugs, and costs. Utilization of L-asparaginase as a therapeutic agent is essential to increase survival of patients. However, costs are elevated and the bacterial forms of the enzyme cause reactions that result in its inhibition by the immune system. Therapeutics alternatives may be searched among eukaryote producers, like fungi. Twelve strains of filamentous fungi were evaluated regarding expression of L-asparaginase activity. The profile of nitrogen assimilation and radial growth were determined for strains which showed higher production ratios. Three media were formulated after selection of the carbon source and carbon/nitrogen ratio that better induced L-asparaginase expression by Penicillium sp. and Fusarium sp. The enzyme activity produced in liquid media reached 8.3 U min.-1 mL-1 (Penicillium sp. T6.2) and 11.4 U min.-1 mL-1 (Fusarium sp.) after 72 hours of cultivation in Bacelar-1 medium. These data show that good producers can be found among fungi, and adjustment of productive processes may offer an alternative to implement eukaryote L-asparaginase production.

O tratamento da leucemia linfoblástica aguda é desafiador devido aos efeitos adversos, à eficácia dos fármacos disponíveis e aos custos envolvidos. A utilização de L-asparaginase como agente terapêutico é fundamental para aumentar a sobrevida do paciente. Porém, seu custo é elevado e as formas bacterianas da enzima causam reações que resultam na sua inibição pelo sistema imune. Alternativas terapêuticas podem ser buscadas entre produtores eucariontes, como os fungos. Doze linhagens de fungos filamentosos foram avaliadas quanto à expressão de atividade de L-asparaginase. O perfil de assimilação de nitrogênio e o crescimento radial foram determinados para as linhagens com maior razão de produção. Três meios foram formulados após a seleção da fonte de carbono e da razão carbono/nitrogênio que melhor induziram a expressão de L-asparaginase por Penicillium sp. e Fusarium sp. A atividade enzimática produzida em meio líquido alcançou 8,32 U min.-1 mL-1 (Penicillium sp. T6.2) e 11,45 U min.-1 mL-1 (Fusarium sp.) após 72 horas de cultivo no meio Bacelar-1. Esses dados mostram que bons produtores podem ser encontrados entre os fungos e que ajustes nos processos produtivos podem oferecer uma alternativa para a implementação da produção de L-asparaginase eucarionte.

Fungal production of the anti-leukemic enzyme L-asparaginase: from screening to medium development / Produção fúngica da enzima antileucêmica L-asparaginase: da triagem ao desenvolvimento de um meio de cultivo

The treatment of acute lymphoblastic leukemia is challenging due to side effects, efficacy of the available drugs, and costs. Utilization of L-asparaginase as a therapeutic agent is essential to increase survival of patients. However, costs are elevated and the bacterial forms of the enzyme cause reactions that result in its inhibition by the immune system. Therapeutics alternatives may be searched among eukaryote producers, like fungi. Twelve strains of filamentous fungi were evaluated regarding expression of L-asparaginase activity. The profile of nitrogen assimilation and radial growth were determined for strains which showed higher production ratios. Three media were formulated after selection of the carbon source and carbon/nitrogen ratio that better induced L-asparaginase expression by Penicillium sp. and Fusarium sp. The enzyme activity produced in liquid media reached 8.3 U min.-1 mL-1 (Penicillium sp. T6.2) and 11.4 U min.-1 mL-1 (Fusarium sp.) after 72 hours of cultivation in Bacelar-1 medium. These data show that good producers can be found among fungi, and adjustment of productive processes may offer an alternative to implement eukaryote L-asparaginase production.(AU)

O tratamento da leucemia linfoblástica aguda é desafiador devido aos efeitos adversos, à eficácia dos fármacos disponíveis e aos custos envolvidos. A utilização de L-asparaginase como agente terapêutico é fundamental para aumentar a sobrevida do paciente. Porém, seu custo é elevado e as formas bacterianas da enzima causam reações que resultam na sua inibição pelo sistema imune. Alternativas terapêuticas podem ser buscadas entre produtores eucariontes, como os fungos. Doze linhagens de fungos filamentosos foram avaliadas quanto à expressão de atividade de L-asparaginase. O perfil de assimilação de nitrogênio e o crescimento radial foram determinados para as linhagens com maior razão de produção. Três meios foram formulados após a seleção da fonte de carbono e da razão carbono/nitrogênio que melhor induziram a expressão de L-asparaginase por Penicillium sp. e Fusarium sp. A atividade enzimática produzida em meio líquido alcançou 8,32 U min.-1 mL-1 (Penicillium sp. T6.2) e 11,45 U min.-1 mL-1 (Fusarium sp.) após 72 horas de cultivo no meio Bacelar-1. Esses dados mostram que bons produtores podem ser encontrados entre os fungos e que ajustes nos processos produtivos podem oferecer uma alternativa para a implementação da produção de L-asparaginase eucarionte.(AU)