Clinical Anslysis of Primary Adrenal NK/T-Cell Lymphoma / 中国实验血液学杂志

OBJECTIVE@#To investigate the clinical characteristics, diagnosis, and treatment of one patient with primary adrenal natural killer/T-cell lymphoma (PANKTCL), and to strengthen the understanding of this rare type of lymphoma.@*METHODS@#The clinical manifestations, diagnosis and treatment process, and prognosis of the patient admitted in our hospital were retrospectively analyzed.@*RESULTS@#Combined with pathology, imaging, bone marrow examination， etc, the patient was diagnosed with PANKTCL (CA stage, stage II; PINK-E score 3, high-risk group). Six cycles of "P-GemOx+VP-16" regimen（gemcitabine 1 g/m2 d1 + oxaliplatin 100 mg/m2 d 1 + etoposide 60 mg/m2 d 2-4 + polyethylene glycol conjugated asparaginase 3 750 IU d 5） was performed, and complete response was assessed in 4 cycles. Maintenance therapy with sintilimab was administered after the completion of chemotherapy. Eight months after the complete response, the patient experienced disease recurrence and underwent a total of four courses of chemotherapy, during which hemophagocytic syndrome occurred. The patient died of disease progression 1 month later.@*CONCLUSION@#PANKTCL is rare, relapses easily, and has a worse prognosis. The choice of the "P-GemOx+VP-16" regimen combined with sintilimab help to improve the survival prognosis of patient with non-upper aerodigestive tract natural killer /T-cell lymphoma.

Efficient production of L-asparaginase in Bacillus licheniformis by optimizing expression elements and host / 生物工程学报

L-asparaginase (L-ASN) is widely applied in the treatment of malignant tumor and low-acrylamide food production, however, the low expression level hampers its application. Heterologous expression is an effective strategy to increase the expression level of target enzymes, and Bacillus is generally used as the host for efficient production of enzymes. In this study, the expression level of L-asparaginase in Bacillus was enhanced through optimization of expression element and host. Firstly, five signal peptides (SPSacC, SPAmyL, SPAprE, SPYwbN and SPWapA) were screened, among which SPSacC showed the best performance, reaching an activity of 157.61 U/mL. Subsequently, four strong promoters (P43, PykzA-P43, PUbay and PbacA) from Bacillus were screened, and tandem promoter PykzA-P43 showed the highest yield of L-asparaginase, which was 52.94% higher than that of control strain. Finally, three Bacillus expression hosts (B. licheniformis Δ0F3 and BL10, B. subtilis WB800) were investigated, and the maximum L-asparaginase activity, 438.3 U/mL, was reached by B. licheniformis BL10, which was an 81.83% increase compared with that of the control. This is also the highest level of L-asparaginase in shake flask reported to date. Taken together, this study constructed a B. licheniformis strain BL10/PykzA-P43-SPSacC-ansZ capable of efficiently producing L-asparaginase, which laid the foundation for industrial production of L-asparaginase.

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.

L-Asparaginase e perspectivas no tratamento da leucemia linfoblástica aguda: revisão de literatura / L-Asparaginase and perspectives in the treatment of leukemia acute lymphoblastic: literature review

Introdução: A L-asparaginase tem sido estudada como alternativa no tratamento da Leucemia Linfoblástica Aguda (LLA) uma vez que possui a capacidade de induzir apoptose em células leucêmicas sem causar danos às células normais. Estudos mostraram benefícios no tratamento da LLA, porém com o risco de desenvolver efeitos adversos. Objetivo: Este trabalho visa apresentar e explicar o histórico da L-asparaginase, desafios enfrentados pelo Brasil, mecanismos de ação que envolvem as formas da enzima e efeitos adversos de sua utilização. Métodos: Foram incluídos neste trabalho 54 artigos na língua portuguesa e inglesa consultados em bancos de artigos como PubMed e SciELO, entre o período de 1953 até 2021. Resultados: A L-asparaginase é uma enzima que converte asparagina em aspartato e amônia, isolada a partir de colônias de Escherichia coli e de Erwinia chrysanthemi, além disso foi polimerizada com polietilenoglicol. O uso de corticosteroides, anti-histamínicos e suplementação vitamínica se mostraram eficientes para amenizar os efeitos adversos. Conclusões: É necessário evitar um desabastecimento de L-Asparaginase no Brasil, principalmente por conta da dificuldade de comercialização e alto custo, mesmo sendo um medicamento presente na lista da Organização Mundial da Saúde, considerado essencial.

Introduction: L-asparaginase has been studied as an alternative in the treatment of Acute Lymphoblastic Leukemia (ALL) since it has the ability to induce apoptosis in leukemic cells without causing damage to normal cells. Studies have shown benefits in the treatment of ALL, but with the risk of developing adverse effects. Objective: This work aims to present and explain the history of L-asparaginase, challenges faced by Brazil, mechanisms of action involving the forms of the enzyme and adverse effects of its use. Methods: 54 articles in Portuguese and English were included in this work, consulted in article banks such as PubMed and SciELO, between the period of 1953 to 2021. Results: L-asparaginase is an enzyme that converts asparagine into aspartate and ammonia, isolated from from Escherichia coli and Erwinia chrysanthemi colonies, it was also polymerized with polyethylene glycol. The use of corticosteroids, antihistamines and vitamin supplementation proved to be efficient in mitigating adverse effects. Conclusions: It is necessary to avoid a shortage of L-Asparaginase in Brazil, mainly due to the difficulty of commercialization and high cost, even though it is a drug present on the World Health Organization list, considered essential.

Desenvolvimento e caracterização de lipossomas peguilados para veiculação de L-asparaginase / Development and characterization of peg-grafted liposomes for lasparaginase encapsulation

A Leucemia Linfoide Aguda (LLA) é um câncer de maior incidência em crianças, e tem a Lasparaginase (ASNase) como fármaco amplamente utilizado no tratamento dos afetados. A ASNase catalisa a hidrólise do aminoácido L-asparagina (Asn), presente na corrente sanguínea, a ausência do aminoácido no meio extracelular leva à morte células leucêmicas, que necessitam deste aminoácido para as funções celulares. Fatores envolvendo a eficiência do tratamento com ASNase como reações adversas e curta meia-vida, principalmente devido ao reconhecimento pelo sistema imune e degradação por proteases, limitam a sua eficácia. A encapsulação da enzima em lipossomas pode conferir proteção à degradação, melhorar seu perfil farmacocinético e diminuir os efeitos adversos, de forma a melhorar o tratamento da LLA sendo este o objetivo desse trabalho. Lipossomas de DOPC (1,2-dioleoil-sn-glicero-3-fosfocolina) e DMPC (1,2-dimiristoil-snglicero-3-fosfocolina) foram desenvolvidos empregando-se o método de hidratação do filme lipídico e diferentes protocolos de preparo contendo ou não diferentes concentrações de 18:0 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polietilenogicol)-2000] (DSPE-PEG). Os lipossomas produzidos foram utilizados para encapsular a ASNase e os sistemas contendo ou não ASNase encapsulada foram caracterizados por espalhamento de luz dinâmico (DLS), potencial zeta, microscopia eletrônica de transmissão (MET) e criomicroscopia de transmissão. Adicionalmente, foram avaliados a taxa de encapsulação e o perfil de permeabilidade das vesículas à L-asparagina. As análises de DLS mostraram que as nanoestruturas formadas empregando-se agitação magnética a partir de sistemas contendo 10% e 20% de DSPE-PEG possuem diâmetro hidrodinâmico menor (~ 25 nm a 60 nm) que os mesmos sistemas sem o fosfolipídio peguilado (~190 nm a 222 nm), demonstrando a relação entre a diminuição do tamanho e o aumento da quantidade de fosfolipídio peguilado e possível formação de estruturas micelares ou bicelares. O emprego de agitação em vórtex para hidratação do filme lipídico, adição do antioxidante -tocoferol e redução da concentração de DSPE-PEG (5% e 10%) levou à formação de sistemas com diâmetro hidrodinâmico maior, sendo esse protocolo e concentrações de PEG definidos como padrão. As análises de MET comprovaram a formação de lipossomas com diâmetro hidrodinâmico semelhante ao observado por DLS; com a utilização da criomicroscopia foi possível observar os lipossomas sem deformações. Os lipossomas de DMPC/DSPE-PEG 10% apresentaram maior permeabilidade à L-asparagina ao longo do tempo e, portanto, poderiam funcionar como nanoreatores, depletando o aminoácido da circulação. Estudos in vitro com células tumorais devem ser realizados e em seguida estudos in vivo, para confirmar este potencial

L-asparaginase (ASNase) is a first-choice drug, combined with other drugs, in therapeutic schemes to treat Acute Lymphoblastic Leukemia (ALL) in children and adolescents. ASNase catalyzes the hydrolysis of L-asparagine (Asn) in the bloodstream; since ALL cells cannot synthesize this amino acid, protein synthesis is impaired leading to leukemic cells death by apoptosis. In spite of its therapeutic importance, treatment with ASNase is associated to side effects, mainly hypersensitivity and immunogenicity. Another drawback refers to degradation by plasma proteases that altogether with immunogenicity shortens the enzyme half-life. Encapsulation of ASNase in liposomes, vesicular nanostructures formed by the self-aggregation of phospholipids, is an attractive alternative that possibly will protect the enzyme from plasma proteases, resulting on better pharmacokinetics profile. In this work, we prepared by thin film hydration liposomal formulations of the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC) containing or not different concentrations of 18:0 1,2-distearoyl-snglycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG), and encapsulated ASNase by electroporation. The systems containing or not ASNase were analyzed by Dynamic Light Scattering, zeta potential and Electron Microscopy. The encapsulation efficiency and vesicles permeability were also evaluated. According to the DLS analysis, the nanostructures formed by film hydration under magnetic stirring employing 10% or 20% DSPE-PEG presented smaller hydrodynamic diameter (~ 25 nm to 60 nm) than the same systems without the pegylated phospholipid (~ 190 nm to 222 nm), demonstrating the relation between size and the amount of pegylated phospholipid that results in formation of micellar or bicellar structures. The protocol was stabilize by hydration of the lipid film under vortex agitation, addition of the antioxidant - tocopherol and reduction of the concentration of DSPE-PEG (5% and 10%), what altogether led to the formation of nanostructures of higher hydrodynamic diameter and monodisperse systems. TEM analyzes confirmed the formation of liposomes with hydrodynamic diameter similar to that observed by DLS; with the use of cryomicroscopy it was possible to observe the liposomes without deformations. Liposomes of DMPC/DSPE-PEG 10% showed permeability to L-asparagine over time and, therefore, could function as nanoreactors, depleting the circulating amino acid

Two-Step Purification of L-Asparaginase from Acrylaway® L

Abstract L-Asparaginase (L-ASNase) is a biopharmaceutical used for acute lymphoblastic leukaemia (ALL) treatment, dramatically increasing the patients' chance of cure. However, its production and distribution in developing countries were disrupted because of its low profitability, which caused great concern among patients. This study evaluates the feasibility of combining fractional precipitation and aqueous two-phase systems (ATPS) to purify L-ASNase from a low-grade product, commercially known as Acrylaway® L. The ATPS purification results were not particularly expressive compared to the two-step purification process composed of ethanol precipitation and gel filtration, which was able to recover the target molecule with a purification factor over 5 fold. Thus, we studied a purification process capable of manufacturing pharmaceutical grade L-ASNase from a commercially available low-grade raw material; however, improvements regarding its throughput must be achieved, and high purity is the first step to apply it as a new biopharmaceutical product. The proposed process could pose as a short-time solution to mitigate its shortage while a cost-effective production plant is being developed.

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.

Allergic reactions to asparaginase: retrospective cohort study in pediatric patients with acute lymphoid leukemia

ABSTRACT Introduction: To assess the frequency of allergic reactions to asparaginase (ASP) and possible risk factors for reactions in a cohort of pediatric patients. Method: The study was performed based on retrospective data from patients under acute lymphoid leukemia treatment in a general university hospital located in southern Brazil. Information on patients who used ASP from 2010 to 2017 was collected. Allergic reactions were identified in electronic medical records. Results: Among the 98 patients included in the study, 16 (16.3 %) experienced an allergic reaction to native l-asparaginase (L-ASP). Of the 22 patients (22.4 %) that received only intravenous (IV) administration of l-ASP, 10 (62.5 %) had allergic reactions, while 48 patients (49 %) received intramuscular (IM) administration and 28 (28.6 %) received IV and IM administrations. The occurrence of allergic reactions differed between the groups (p < 0.001), and IV administration was associated with allergic reactions. Association was also observed between the severity of the reaction and the route of administration, with the IM route associated with grade 2 and IV route associated with grade 3. Occurrence of allergic reactions was higher when the commercial formulation of l-ASP, Leuginase®, was used (p = 0.0009 in the analysis per patient and p = 0.0003 in the analysis per administration). Conclusions: The IV administration and commercial Leuginase® presentation were associated with more allergic reactions in the study population, which corroborates the findings in the literature. The IV route was also associated with higher severity of reactions in the present study.

Effects of site-directed PEGylation on L-asparaginase thermostability / Efeitos da PEGuilação sítio-dirigida na termoestabilidade da L-asparaginase

The enzyme L-asparaginase (ASNase) is broadly applied as a drug to treat acute lymphoblastic leukemia, as well as in the food industry to avoid acrylamide formation in baked and fried food. In the present work, ASNase was covalently attached to polyethylene glycol (PEG) of different molecular weights (ASNase-PEG-5, ASNase-PEG-10, ASNase-PEG-20, and ASNase-PEG-40) at the N-terminal portion (monoPEGylation). Native and PEGylated forms were analyzed regarding thermodynamics and thermostability based on enzyme activity measurements. ASNase (native and PEGylated) presented maximum activity at 40 °C and denaturation followed a first-order kinetics. Based on these results, the activation energy for denaturation (E*d) was estimated and higher values were observed for PEGylated forms compared to the native ASNase, highlighting the ASNase-PEG10 with a 4.24-fold increase (48.85 kJ.mol-1) in comparison to the native form (11.52 kJ.mol-1). The enzymes were evaluated by residual activity over time (21 days) under different storage temperatures (4 and 37 °C) and the PEGylated conjugates remained stable after the 21 days. Thermodynamic parameters like enthalpy (ΔH‡), entropy (ΔS‡) and Gibbs free energy (ΔG‡) of ASNase (native and PEGylated) irreversible denaturation were also investigated. Higher - and positive - values of Gibbs free energy were found for the PEGylated conjugates (61.21 a 63.45 kJ.mol-1), indicating that the process of denaturation was not spontaneous. Enthalpy also was higher for PEGylated conjugates (18.84 a 46.08 kJ.mol-1), demonstrating the protective role of PEGylation. As for entropy, the negative values were more elevated for native ASNase (-0.149 J/mol.K), pointing out that the denaturation process enhanced the randomness and aggregation of the system, which was observed by circular dichroism. Thus, PEGylation proved its potential to increase ASNase thermostability

A enzima L-asparaginase (ASNase) é amplamente usada como medicamento para tratamento da leucemia linfoblástica aguda, bem como na indústria de alimentos para evitar a formação de acrilamida em alimentos cozidos e fritos. No presente trabalho, ASNase foi covalentemente ligada ao polímero poli(etilenoglicol) (PEG) de diferentes massas moleculares (ASNase-PEG-5, ASNase-PEG- 10, ASNase-PEG-20, and ASNase-PEG-40) na região N-terminal (monoPEGuilação) a fim de se estudar os efeitos da PEGuilação na termoestabilidade da enzima. As formas PEGuiladas e nativa foram analisadas em relação à termodinâmica e termoestabilidade a partir de atividade enzimática. A ASNase (nativa e PEGuilada) apresentou atividade máxima a 40 °C e a desnaturação ocorreu por cinética de primeira ordem. Com base nesses resultados, a energia de ativação para desnaturação (E*d) foi estimada e maiores valores foram observados para as formas PEGuiladas em comparação à enzima nativa, destacando-se a ASNase-PEG10 com aumento de 4.24 vezes (48.85 kJ.mol-1) em comparação com a forma nativa in (11.52 kJ.mol mol-1). As enzimas foram avaliadas por sua atividade residual ao longo do tempo em diferentes temperaturas de armazenamento (4 e 37 °C) e os conjugados PEGuilados mostraram-se mais estáveis após os 21 dias de ensaio. Parâmetros termodinâmicos como entalpia (ΔH‡) de desnaturação irreversível foram analisados. Valores maiores - e ), entropia (ΔS‡) de desnaturação irreversível foram analisados. Valores maiores - e ) e energia livre de Gibbs (ΔG‡) de desnaturação irreversível foram analisados. Valores maiores - e positivos - da energia livre de Gibbs foram encontrados para os conjugados PEGuilados (61.21 a 63.45 kJ.mol-1), indicando que o processo de desnaturação não ocorreu de forma espontânea. A entalpia também foi maior para os conjugados PEGuilados (18.84 a 46.08 kJ.mol-1), demonstrando o efeito protetivo da PEGuilação. Já para a entropia, os valores negativos foram mais elevados para a ASNase nativa (-0.149 J/mol.K), apontando que o processo de desnaturação aumentou a aleatoriedade e agregação do sistema, o que foi confirmado pelo dicroísmo circular. Dessa forma, a PEGuilação revelou o seu potencial de aumento de termoestabilidade para a ASNase

Análise Técnico-Econômica para a produção de biofármacos de L-asparaginase / Technical-Economic analysis for the production of L-asparaginase biopharmaceuticals

A enzima L-asparaginase é comumente utilizada como biofármaco para o tratamento da Leucemia Linfoblástica Aguda e possui altas taxas de cura com o medicamento disponível no mercado. Atualmente a aquisição deste biofármaco é fruto integral de importação, não sendo realizada produção nacional, muito embora existam grupos de pesquisas nacionais que trabalham em pesquisas e no desenvolvimento de biofármacos alternativos da L-asparaginase. Assim, a presente dissertação tem como objetivo realizar análises técnico-econômicas para avaliar a viabilidade de implementação industrial de bioprocessos para a produção da L-asparaginase do tipo Erwinase PEGuilada e não PEGuilada, que foram previamente desenvolvidos na FCF-USP. As análises técnico-econômicas foram conduzidas por meio do software SuperPro Design® (Intelligen, Inc.) e permitiram adaptar o processo laboratorial para um processo piloto e possibilitaram estimar os valores de custo de produção unitário (Unity Cost of Production - UPC) de US$ 12,37/mg e US$ 3,46/mg para a L-asparaginase monoPEGuilada e nativa obtida por processo similar, respectivamente. O custo unitário de produção para a enzima peguilada foi, portanto, estimado em cerca de 4 vezes o mesmo custo para a produção da enzima peguilada, sendo tal aumento de custo devido às operações de peguilação, já que ambas as plantas foram mantidas nas mesmas dimensões. Ainda, foram obtidos indicadores econômicos, que indicam a atratividade do processo desenvolvido, muito embora tenham sido identificados diversos gargalos de processo e fatores a serem otimizados e melhorados de forma a tornar o processo mais atrativo sob os pontos de vista técnico e econômico. Em uma análise de sensibilidade preliminar um aumento factível da densidade celular já mostra que é possível reduzir em mais de 30% o UPC. De toda forma, ainda que não otimizado, o processo apresentou valores e dados compatíveis com os biofármacos de L-asparaginase já disponíveis no mercado

The enzyme L-asparaginase is commonly used as a biopharmaceutical in the treatment of Acute Lymphoblastic Leukemia, presenting high cure rates with the formulations available on the market. Nowadays, the acquisition of this biopharmaceutical is only from importation, given that there is no national production being carried out, although there are national research groups working on research and development of alternative L-asparaginase biopharmaceuticals. Thus, this project aims at carrying out technical-economic analyzes to evaluate the viability of industrial implementation of bioprocesses for the production of L-asparaginase of the PEGylated and non-PEGylated Erwinase type previously developed at FCF-USP. The technical-economic analyzes, conducted by means of the software SuperPro Design® (Intelligen, Inc.), allowed to adapt the laboratory process to a pilot process and made it possible to estimate the unit cost of production (UPC) values of US $ 12.37 / mg and US $ 3.56 / mg for monoPEGylated L-asparaginase and bare obtained by similar process, respectively. The unit cost of production for the pegylated enzyme was, therefore, estimated at about 4 times the same cost for the production of the pegylated enzyme, such an increase in cost due to pegylation operations, since both plants were maintained in the same dimensions. Moreover, economic indicators were obtained, which indicate the attractiveness of the developed process. However, several process bottlenecks and factors to be optimized and improved were identified to make the process more attractive from the technical and economic point of view. In a preliminary sensitivity analysis, a feasible increase in cell density already shows that it is possible to reduce UPC by more than 30%. Accordingly, although not optimized, the process presented values and data compatible with the L-asparaginase biopharmaceuticals already available on the market

Estudo da estabilidade e peguilação de L-asparaginase em soluções aquosas de líquidos iônicos de imidazólio / Study of the stability and pegylation of L-asparaginase in aqueous imidazole ionic liquid solutions

A enzima L-asparaginase de Escherichia coli (ASNase) é um biofármaco indicado para o tratamento de leucemia linfoblástica aguda, mas que pode causar reações de hipersensibilidade nos pacientes tratados. Na tentativa de amenizar esse efeito, foi desenvolvida a PEG-ASNase (enzima conjugada com polietilenoglicol) que apresenta a vantagem de ser menos imunogênica e ter maior meia-vida biológica. Mais recentemente, novas abordagens têm sido desenvolvidas visando aprimorar os processos de PEGuilação por meio de reações sítio dirigidas, por exemplo N-terminal, a fim de promover maior similaridade lote a lote e controle das características farmacocinéticas e farmacodinâmicas do biofármaco. Porém, existe ainda uma limitação associada à hidrólise do PEG reativo, desta forma surge a necessidade de procurar solventes alternativos para a PEGuilação que permitam manter a estabilidade das proteínas, aumentar o rendimento de PEGuilação e a estabilidade do PEG reativo. Nesse trabalho, líquidos iônicos foram investigados como solventes alternativos para a peguilação N-terminal de PEG-ASNase. Para tal, a estabilidade de ASNase em Lis foi investigada em LIs da família metil-imidazol, analisando a influência do aumento da cadeia alquílica e de diferentes ânions. A estabilidade da ASNase é favorecida quando em contato com Lis relativamente hidrofóbicos ([C2mim]Cl, [C4mim]Cl e [C6mim]Cl), mas sua a atividade é prejudicada quando o LI é muito polar, como o [C4mim][(CH3)2PO4] ou anfifílico como o [C12mim]Cl. Apesar de seu efeito desnaturante, o [C4mim][(CH3)2PO4] resultou no maior rendimento da reação de PEGuilação da ASNase (56%) quando empregado a 75% e a reação realizada em 10 min. O [C4mim]Cl resultou em rendimento semelhante ao tampão fosfato (~ 49%), mas ambos os LIs reduziram a poliPEGuilação. Portanto, os Lis [C4mim]Cl e [C4mim][(CH3)2PO4] fornecem uma alternativa viável à reação de PEGuilação pela redução na formação de espécies poliPEGuiladas, o que facilitaria os processos de purificação e permitiria maior controle lote a lote da reação, bem como pelo aumento do rendimento da reação no caso do [C4mim][(CH3)2PO4]

Escherichia coli L-asparaginase enzyme (ASNase) is a biopharmaceutical indicated for the treatment of acute lymphoblastic leukemia, but may cause hypersensitivity in the patients used. In an attempt to alleviate this effect, PEG-ASNase (polyethylene glycol conjugated enzyme) was developed, which has the advantage of being less immunogenic and having a longer biological half-life. More recently, new approaches have been applied to improve PEGylation processes through targeted sites, for example N-terminal, in order to promote greater similarity to the batch and control of the pharmacokinetic and pharmacodynamic characteristics of the biopharmaceutical. However, there is still a limitation associated with reactive PEG hydrolysis, thus increasing the need to look for alternative PEGylation solvents to maintain protein stability, increase PEGylation yield and use reactive PEG. In this work, ions were investigated as alternative solvents for the N-terminal PEG-ASNase. For example, a stability of ASNase in ILs was investigated in imidazole ILs by analyzing the influence of increased alkyl chain and different anions. ASNase stability is enhanced when in contact with relatively hydrophobic ILs ([C2min]Cl, [C4min]Cl and [C6min]Cl), but its activity is impaired when very polar ILs such as [C4min][(CH3)2PO4] or amphiphilic as [C12mim]Cl. Despite its denaturing effect, [C4min][(CH3)2PO4] resulted in higher yield of ASNase PEGylation reaction (56%) when employed at 75% and reaction performed in 10 min. [C4min]Cl yielded similar phosphate buffer yield (~ 49%), but both ILs reduced polyPEGylation. Therefore, [C4min]Cl and [C4min][(CH3)2PO4] Ils may use a viable alternative to the PEGylation reaction and reduce the formation of polyPEGylated species, or that facilitate purification processes and allow for greater batch use of the solution, as well as increased reaction yield in the case of [C4min][(CH3)2PO4]

Cetoacidose diabética e pancreatite secundárias à peg-asparaginase / Diabetic ketoacidosis and pancreatitis secondary to peg-asparaginase

RESUMO: Objetivos: Apresentar um caso raro de cetoacidose diabética (CAD) e pancreatite secundários ao uso de PEG-asparaginase em paciente pediátrico em tratamento para leucemia linfoblástica aguda (LLA) e alertar quanto aos sinais que remetem a esses diagnósticos. Descrição do caso: Adolescente do sexo feminino, 10 anos e 11 meses, em tratamento para LLA e uso prévio de PEG-asparaginase há seis dias da internação, admitida com choque hipotensivo grave e encaminhada à Unidade de Terapia Intensiva. Inicialmente o quadro foi interpretado como choque séptico. Em seguida a anamnese detalhada e os exames laboratoriais direcionaram para os diagnósticos de CAD e pancreatite, iniciando-se as intervenções específicas. Recebe alta hospitalar após 30 dias, sem necessidade de insulinoterapia, mas com reposição de enzimas pancreáticas. Comentários: Geralmente, às crianças com LLA gravemente enfermos e leucopênicos, atribui-se apenas o diagnóstico de sepse, que é um diagnóstico prioritário. Entretanto, no grupo em uso de PEG-asparaginase, o pediatra emergencista deve estar alerta ao raciocínio diferencial envolvendo CAD e pancreatite, o que pode ser bem difícil inicialmente. O alerta dos diagnósticos diferenciais do choque séptico, mesmo que raros, na assistência a pacientes oncológicos pediátricos, além da correta e pronta identificação do quadro e seu manejo apropriado, correlacionam-se diretamente ao sucesso terapêutico e, em algumas situações, à sobrevivência do paciente. (AU)

ABSTRACT: Objectives: We present a rare case of diabetic ketoacidosis (DKA) and pancreatitis secondary to the use of PEG-asparaginase in a pediatric patient being treated for acute lymphoblastic leukemia (ALL) and draw attention to the signs that refer to these diagnoses. Case description: A female adolescent, aged 10 years and 11 months, undergoing treatment for ALL, used PEG-asparaginase for 6 days prior to admission. She was hospitalized due to severe hypotensive shock and was then referred to the intensive care unit. Initially, the clinical condition was interpreted as septic shock. However, detailed anamnesis and results of laboratory tests led to the diagnoses of DKA and pancreatitis; hence, appropriate interventions were initiated. She was discharged after 30 days without the need for insulin therapy but received pancreatic enzyme replacement therapy. Comments: Generally, diagnosing severely ill and leukopenic children with ALL is only attributed to sepsis, which is a priority diagnosis. However, in the group treated with PEG-asparaginase, the pediatric emergency specialist should consider differential reasoning in patients with DKA and pancreatitis, which can be quite difficult to assess initially. Alertness towards the differential diagnoses of septic shock, although rare, in the care of pediatric oncology patients, in addition to the correct and prompt identification of the condition and provision of appropriate management, directly correlates with treatment success and, in some situations, the improvement in patient's survival. (AU)

Molecular modification and highly efficient expression of L-asparaginase from Rhizomucor miehei / 生物工程学报

L-asparaginase hydrolyzes L-asparagine to produce L-aspartic acid and ammonia. It is widely distributed in microorganisms, plants and serum of some rodents, and has important applications in the pharmaceutical and food industries. However, the poor thermal stability, low catalytic efficiency and low yield hampered the further application of L-asparaginase. In this paper, rational design and 5' untranslated region (5'UTR) design strategies were used to increase the specific enzyme activity and protein expression of L-asparaginase derived from Rhizomucor miehei (RmAsnase). The results showed that among the six mutants constructed through homology modeling combined with sequence alignment, the specific enzyme activity of the mutant A344E was 1.5 times higher than the wild type. Subsequently, a food-safe strain Bacillus subtilis 168/pMA5-A344E was constructed, and the UTR strategy was used for the construction of recombinant strain B. subtilis 168/pMA5 UTR-A344E. The enzyme activity of B. subtilis 168/pMA5 UTR-A344E was 7.2 times higher than that of B. subtilis 168/pMA5-A344E. The recombinant strain B. subtilis 168/pMA5 UTR-A344E was scaled up in 5 L fermenter, and the final yield of L-asparaginase was 489.1 U/mL, showing great potential for industrial application.

Clinical Characteristics and Prognostic Analysis of Extranodal NK/T-Cell Lymphoma Patients Treated with Pegaspargase Based Chemotherapy / 中国实验血液学杂志

OBJECTIVE@#To investigate the clinical characteristics of patients with extranodal NK/T-cell lymphoma (ENKL), and to analyze the factors that affecting the survival and prognostic of patients treated with pegaspargase based chemotherapy.@*METHODS@#The clinical data of 61 ENKL patients treated in Peking Union Medical College Hospital from January 2015 to June 2019 were enrolled and retrospectively analyzed. The clinical characteristics, survival rate and influencing factors of prognostic in patients were investigated.@*RESULTS@#The male and female ratio in the whole group was 2.8∶1. The median age was 46 years old (range, 17-67 years old). 30 patients were in stage I/II, while 31 patients were in stage III/IV. The ratio of nasal and non-nasal type was 4.1∶1. The common sites of extranodal involvement were skin and subcutaneous tissue (26.2%), liver (14.8%), lung (13.1%) and gastrointestinal tract (13.1%). 9.8% of patients showed central nervous system involvement and 11.5% showed bone marrow involvement. The median follow-up time was 22 months (range, 1-53 months). The 2-year PFS and OS rates of patients in the whole group were 51.6% and 53.2%, respectively. The 2-year OS rate of patients at stage I/II was 87.5%, while that of patients at stage III/IV was only 21.2%, the difference showed statistically significant (P60 years old and Ann Arbor stage III-IV were the independent adverse factors that affecting the prognosis of PFS and OS (HR=3.681, 95% CI 1.322-10.250; HR=4.611, 95% CI 1.118-19.009).@*CONCLUSION@#The survival of ENKL patients has been significantly improved by pegaspargase based chemotherapy. Patients with stage I/II disease have achieved a relatively good 2-year OS rate of 87.5%, but the prognosis of stage III/IV and non-nasal type patients are still poor. Age>60 years old and Ann Arbor stage III/IV are independent adverse prognostic factors for ENKL patients.

Asparaginase: an old drug with new questions

ABSTRACT The long-term outcome of acute lymphoblastic leukemia has improved dramatically due to the development of more effective treatment strategies. L-asparaginase (ASNase) is one of the main drugs used and causes death of leukemic cells by systematically depleting the non-essential amino acid asparagine. Three main types of ASNase have been used so far: native ASNase derived from Escherichia coli, an enzyme isolated from Erwinia chrysanthemi and a pegylated form of the native E. coli ASNase, the ASNase PEG. Hypersensitivity reactions are the main complication related to this drug. Although clinical allergies may be important, a major concern is that antibodies produced in response to ASNase may cause rapid inactivation of ASNase, leading to a worse prognosis. This reaction is commonly referred to as "silent hypersensitivity" or "silent inactivation". We are able to analyze hypersensitivity and inactivation processes by the measurement of the ASNase activity. The ability to individualize the ASNase therapy in patients, adjusting the dose or switching patients with silent inactivation to an alternate ASNase preparation may help improve outcomes in those patients. This review article aims to describe the pathophysiology of the inactivation process, how to diagnose it and finally how to manage it.

PEG-asparaginase and native Escherichia coli L-asparaginase in acute lymphoblastic leukemia in children and adolescents: a systematic review

Abstract Introduction Acute lymphoblastic leukemia (ALL) is the cancer with the highest incidence in childhood and adolescence, and pharmacotherapy is the primary form of treatment. Objective and methods A systematic review of the efficacy and safety of polyethylene glycol (PEG)-asparaginase in acute lymphoblastic leukemia therapy in children and adolescents was conducted to compare it with native Escherichia coli L-asparaginase. PubMed, Web of Science, Science Direct, Cochrane Library, Scopus, LILACS (Latin American and Caribbean Health Sciences Literature) and EMBASE databases were selected. The following outcomes were analyzed: complete remission of the disease, event-free survival, overall survival, anti-asparaginase antibody level, hypersensitivity reactions, asparaginase and asparagine serum levels, number of postdiagnosis events, and overall mortality. Five randomized controlled trials were included. Analysis of the quality of evidence and risk of bias was performed using the Cochrane recommendation tool and the GRADE system. Results The assessment results suggest that the level of certainty on the technology addressed is relatively weak from a methodological point of view. Evidence is insufficient to assess the effects on health outcomes because of the limited number and power of studies and important flaws in their design or conduct in classifying PEG-asparaginase as a superior drug or not, in the pharmacotherapy of ALL in children and adolescents. PEG-asparaginase can be used as a substitute for native E. coli L-asparaginase, demonstrating similar efficacy and safety. Conclusion The study may help decision-makers in the public health system to offer a more in-depth judgment on the therapeutic alternatives used to treat this neoplasm in children and adolescents.

L-asparaginase de Erwinia chrysanthemi: expressão proteica livre de células e bioconjugação a bacteriófagos / L-asparaginase from Erwinia chrysanthemi: cell-free protein expression and bioconjugation to bacteriophages

A L-Asparaginase (L-ASNase) de Erwinia chrysathemi (ErA) é uma enzima amplamente utilizada para o tratamento da leucemia linfoblástica aguda (LLA). Embora o seu uso como segunda linha de tratamento para a LLA tenha proporcionado consideráveis benefícios clínicos, reações de hipersensibilidade e rápida depuração plasmática ainda são problemas recorrentes. Ademais, extensivos e custosos processos de produção da ErA são necessários para a obtenção da enzima pura. Com base nesses problemas, o presente trabalho propõe (1) o estudo de viabilidade de expressão da ErA em um sistema de síntese proteica livre de células (SPLC) e (2) a conjugação da proteína em bacteriófagos como ferramenta alternativa para o isolamento e monitoramento da depuração plasmática da ErA. Foram utilizados extratos celulares de Escherichia coli suplementados com solução energética contendo creatina fosfato (CP) como fonte de energia para síntese in vitro de ErA. Para conjugação da ErA a bacteriófagos, o sistema SpyTag/SpyCatcher foi implementado: SpyCatcher foi fusionado à porção N-terminal da ErA e bacteriófagos filamentosos da linhagem M13 e fd foram modificados de modo a expressar SpyTag nas proteínas de capsídeo pIII e pVIII, respectivamente. Em relação ao primeiro objetivo, o sistema de SPLC foi capaz de expressar a ErA com atividade. A proteína foi expressa na fração solúvel e apresentou atividade enzimática significativamente superior em relação à reação controle (7,07 ± 0,68 U/mL vs. 1,83 ± 0,14 U/mL). Tempo necessário para obtenção do extrato celular foi reduzido de 45 para 26 hrs, e sete componentes da solução energética foram removidos da composição original sem implicações negativas na eficiência de expressão da ErA, simplificando desta forma o processo de SPLC. Em relação ao segundo objetivo, ErA fusionada à SpyCatcher (SpyCatcher_ErA) foi conjugada com êxito em bacteriófagos capazes de expressar SpyTag fusionadas na porção N-terminal das proteínas pIII (SpyTag_pIII) e pVIII (SpyTag_pVIII). A porcentagem de formação dos conjugados entre SpyCatcher_ErA e SpyTag_pIII ((ErA)5-pIII) foi de 6% enquanto formação dos conjugados entre SpyCatcher_ErA e SpyTag_pVIII ((ErA)50-pVIII) foi de 46%, valores estes confirmados por atividade enzimática. Solução contendo conjugados foram injetados em camundongos e sequenciados/titulados com êxito. Não houve diferença de depuração plasmática entre (ErA)5-pIII e bacteriófago controle, mas houve maior taxa de eliminação de (ErA)50-pVIII em relação ao mesmo bacteriófago não conjugado à SpyCatcher_ErA. Os resultados aqui apresentados confirmam ser possível expressar ErA com atividade biológica em sistemas de SPLC. Além disso, o sistema de conjugação da ErA a bacteriófagos aqui desenvolvido foi capaz de monitorar a concentração de ErA presente na circulação em função do tempo, tornando-se uma potencial plataforma de desenvolvimento de novas proteoformas da ErA com características clínicas melhoradas

L-Asparaginase (L-ASNase) from Erwinia chrysanthemi (ErA) is a widely used enzyme for treatment of acute lymphoblastic leukemia (ALL). Although its use as a second-line treatment has provided significant clinical benefits, hypersensitivity reactions and a fast clearance rate are recurring L-ASNase-related problems. In addition, extensive and costly production processes are required for the manufacturing of pure ErA. Based on these drawbacks, this current work proposes (1) the study of the use of a cell-free protein synthesis (CFPS) system as a viable platform for the synthesis of ErA and (2) the conjugation of the protein on bacteriophages as an alternative tool for the isolation and monitoring of ErA clearance. Escherichia coli-derived cell extracts supplemented with a creatine phosphate-based energy solution were used to synthesize ErA in vitro. To conjugate ErA on bacteriophages, the SpyTag/SpyCatcher system was implemented: SpyCatcher was fused to the N-terminus of the ErA while filamentous phage strains M13 and fd were engineered in order to display SpyTag on their pIII and pVIII capsid proteins, respectively. Regarding the first goal, the CFPS system was able to express an active ErA. The protein was expressed in the soluble fraction and there presented a significant higher enzymatic activity compared to the control reaction (7.07 ± 0.68 U/mL vs. 1.83 ± 0.14 U/mL). Time required to obtain the cell extract was reduced from 45 to 26 hours, and seven energy solution reagents were removed from the original solution without compromising the efficiency of ErA expression, thus simplifying the CFPS process. With respect to the second goal, ErA fused to SpyCatcher (SpyCatcher_ErA) was sucessfully conjugated on bacteriophages capable of displaying SpyTag fused to the Nterminus of the pIII (SpyTag_pIII) or pVIII (SpyTag_pVIII) proteins. Percentage of conjugate formation between SpyCatcher_ErA and SpyTag_pIII (ErA)5-pIII was 6% whereas conjugate formation between SpyCatcher_ErA and SpyTag_pVIII (ErA)50-pVIII was 46%, values that were confirmed by enzymatic activity. Sample containing conjugates were injected into mice and sucessfully sequenced/titrated. No clearance differences were observed between (ErA)5- pIII and a control bacteriophage, but a higher clearance rate was observed for (ErA)50-pVIII compared to SpyTag_VIII non conjugated to SpyCatcher_ErA. The results here presented confirm the expression of a biologically active ErA from a CFPS system. Besides, the development of a conjugation system capable of linking ErA to bacteriophages could be used as a means to monitor the ErA concentration in the blood as a function of time and also as a potential platform to be used in the development of novel ErA proteoforms with improved clinical properties

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.

Produção de L-asparaginase pela levedura Leucosporidium muscorum CRM 1648 isolada de sedimento marinho coletado na Península Antártica / L-asparaginase production by the yeast Leucosporidium muscorum CRM 1648 isolated from marine sediments collected in Antarctic Peninsula

L-asparaginase (L-ASNase) é uma enzima com propriedades interessantes para a indústria médica, farmacêutica e de alimentos, que tem recebido atenção especial, inclusive no Brasil, por fazer parte do protocolo de tratamento de distúrbios linfoproliferativos, como a leucemia linfoblástica aguda (LLA). No mercado desde a década de 1970, as enzimas de origem bacteriana enfrentam algumas limitações por provocarem reações adversas graves em quase 80% dos pacientes em tratamento. Nesse contexto, L-ASNases provenientes de leveduras se destacam como alternativa, por serem mais próximas às congêneres humanas. A Antártica ainda é um ambiente pouco explorado, com grande diversidade de microrganismos com potencial para a produção de moléculas biológicas de interesse industrial. Nesse contexto, 150 leveduras isoladas de amostras de sedimento marinho coletadas na Península Antártica como parte do projeto MICROSFERA (PROANTAR/CNPq) foram avaliadas para a produção de L-ASNase. A triagem resultou em 9 isolados produtores, dos quais 7 pertencem ao gênero Leucosporidium. A linhagem L. muscorum CRM 1648 foi a que produziu mais enzima (540 U.L-1), com maior produtividade (5,6 U.L-1.h-1) e, por isso, foi alvo deste estudo. A análise univariada de fontes de carbono e nitrogênio indicou maior crescimento desse microrganismo e produção de L-ASNase em meio CD com extrato de levedura, prolina e sacarose. Ureia, cloreto de amônio e sulfato de amônio resultaram em baixa ou nenhuma produção da enzima, sugerindo que a metabolização de fontes de nitrogênio por essa linhagem está sob a influência do fenômeno de repressão catabólica pelo nitrogênio (RCN). Dois delineamentos experimentais do tipo fatorial completo resultaram em um aumento de 10 vezes na produção e produtividade da enzima (4582,5 U.L-1 e 63,6 U.L-1.h-1, respectivamente). A análise univariada da concentração inicial de inóculo (X0), pH inicial do meio, temperatura e adição de água do mar mostrou que a melhor condição para a produção foi: pH = 5,5 ou 6,5, cultivo a 15°C com adição de água do mar (25-50% m/v). A variável X0 não foi significativa nas concentrações avaliadas. Cultivos em biorreator (batelada) foram conduzidos em quatro diferentes níveis de oxigênio dissolvido (OD): (1) OD não controlado e abaixo de 20%, (2) OD não controlado e acima de 20%, (3) OD controlado em 80% e (4) OD controlado em 20%. Os resultados mostraram que OD é fator limitante para o crescimento de L. muscorum CRM 1648 e produção de L-ASNase por essa levedura e deve ser mantido acima de 35% para maior produção da enzima.Neste trabalho, a composição do meio e condições de cultivo foram estabelecidas para favorecer a produção de uma nova L-ASNase livre de atividade glutaminásica por levedura adaptada ao frio, abrindo espaço para novos estudos acerca de seu potencial antileucêmico e possível uso como alternativa às enzimas já existentes no mercado no tratamento de LLA

L-asparaginase (L-ASNase) is an enzyme with interesting properties for medical, pharmaceutical and food industry, which has received special consideration, especially in Brazil, for being part of lymphoproliferative disorders treatment, such as acute lymphoblastic leukemia (ALL). Bacterial enzymes are on the market since the 1970s and face some limitations related to theirserious adverse reactions that reach almost 80% of all patients in treatment. In this context, L-ASNases from yeasts are highlighted as important alternative to bacterial enzymes, due to the closerphylogeny to human congeners. Antarctic environment has much to be explored, with a vast diversity of microorganisms with potential to produce biomolecules with industrial interest. A total of 150 yeasts isolated from Antarctic marine sediments as part of MICROSFERA project (PROANTAR/CNPq) were evaluated for L-ASNase production. The screening resulted in 9 producers, 7 species from the genus Leucosporidium. L. muscorum CRM 1648 was the strain that yielded the highest L-ASNase activity (540 U.L-1) and volumetric productivity (5.6 U.L-1.h-1). Carbon and Nitrogen sources were evaluated by a method of one-factor at a time (OFAT). From the gather results, sucrose, yeast extract and proline resulted in a maximal growth and highest enzyme production.The absence or low production of L-ASNase in medium with urea, ammonium chloride and ammonium sulfate suggests the presence of nitrogen catabolic repression (NCR). Carbon and nitrogen concentration were evaluated by full factorial design and yielded about ten times higher enzyme and volumetric productivity (4582.5 U.L-1 and 63.6 U.L-1.h-1, respectively). Initial inoculum concentration (X0), initial pH, temperature and concentration of seawater in the culture were evaluated by OFAT analysis and the best condition for L-ASNase production was: pH = 5.5 or 6.5, at 15 °C with addition of seawater (25-50 wt%). X0 was not considered a significant variable. Bioreactor assays (in batch regime) were performed in four different dissolved oxygen (DO) levels: (1) without DO control (DO remained under 20%), (2) without DO control (DO remained above 20%), (3) DO controlled at 80%, and (4) DO controlled at 20%.The results showed that DO is a key factor for growth of L. muscorum CRM 1648 and production of L-ASNase by this yeast and should be maintained above 35% for higher production of this enzyme.At this work, the medium and culture conditions were established to support the production of a novel glutaminase-free L-ASNase by a cold adapted yeast, opening a new path for further studies regarding its antileukemic potential and possible use as an alternative for ALL treatment

Expressão da L-asparaginase II de Saccharomyces cerevisiae recombinante extracelular com glicosilação humanizada em Pichia pastoris / Extracellular expression of Saccharomyces cerevisiae's L-asparaginase II in Pichia pastoris with humanized glycosylation

L-asparaginase é um inibidor eficiente do crescimento tumoral, usado em sessões de quimioterapia contra a Leucemia Linfoblástica Aguda (LLA), resultando na remissão completa da doença em 90% dos pacientes tratados. A L-asparaginase II de Saccharomyces cerevisiae (ScASNaseII) tem alto potencial de superar os efeitos adversos da L-asparaginase de bactéria, porém sua produção endógena resulta em uma proteína hipermanosilada e, consequentemente, imunogênica. A cepa de Pichia pastoris Glycoswitch tem a maquinaria para expressar e secretar altas quantidades de enzima com glicosilação humanizada. Nesse trabalho, descrevemos o processo genético para expressar a ScASNaseII no meio extracelular pela P. pastoris Glycoswitch, e também os parâmetros bioquímicos, perfil cinético, citotoxicidade contra células leucêmicas e a interferência da glicosilação na atividade da enzima obtida. Nossos dados mostram que a cepa aplicada foi capaz de expressar ScASNaseII no meio extracelular passível de purificação de proteínas contaminantes com apenas um passo cromatográfico. A atividade específica para asparagina foi 218,2 UI/mg e a atividade glutaminásica representou 3,1% da atividade asparaginásica. Os parâmetros cinéticos foram KM = 120,5 µM e a eficiência catalítica de 3,8 x 105 M-1s-1. Análises por meio de gel nativo sugerem uma conformação tetramérica de aproximadamente 150 kDa. Essa é uma nova estratégia de produzir essa enzima de forma extracelular, com mais facilidade de purificação e com melhores propriedades biotecnológicas

L-asparaginase is an efficient inhibitor of tumor development, used in chemotherapy sessions against acute lymphoblastic leukemia (ALL) tumor cell; its use results in 90% complete remission of the disease in treated patients. Saccharomyces cerevisiae's L-asparaginase II (ScASNaseII) has a high potential to overcome the side effects of bacteria L-asparaginase, but the endogenous production of it results in hypermannosylated immunogenic enzyme. However, Pichia pastoris Glycoswitch strain has the machinery to express and secrete high quantity of the enzyme and with humanized glycosylation. Here we describe the genetic process to acquire the ScASNaseII in the extracellular medium expressed by P. pastoris Glycoswitch, and the biochemical properties of the resultant enzyme, kinetic profile, cytotoxicity against ALL cell line and the interference of glycosylation in its activity. Our data show that the strain employed is able to express extracellular asparaginase active and possible to be purified of contaminant proteins using a single chromatographic step. The specific activity using asparagine was 218.2 IU.mg-1 and the glutaminase activity represents 3.1% of its asparaginase activity. The kinetics parameters were KM=120.5 µM and a catalytic efficiency of 3.8x105 M-1s-1. The Native-PAGE suggested a tetrameric protein conformation, with approximately 150 kDa. This is a novel strategy to produce this enzyme extracellularly, easier to purify and with better biotechnological properties