A rapid, efficient and sensitive plate assay for detection and screening of<scp>l</scp>-asparaginase-producing microorganisms

Mahajan, Richi V.; Saran, Saurabh; Saxena, Rajendra Kumar; Srivastava, Ayush

doi:10.1111/1574-6968.12100

Cited by 58 publications

(41 citation statements)

References 9 publications

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“…Plates/broth containing BTB were yellow at lower pH value and changed to dark blue at higher pH value. Hence, a dense dark blue color is produced if microorganism produces L-asparaginase [25]. In control plate without dye, organism growth was observed with no change in media color.…”

Section: Isolation and Characterization Of Actinomycetes And Screeninmentioning

confidence: 98%

“…The isolates were maintained on SC agar and sub-cultured periodically. Isolates were screened for production of L-asparaginase using the method as described by Mahajan et al [25]. Modified M-9 medium (composition: Na 2 HPO 4 -0.6%; KH 2 PO 4 -0.3%, NaCl-0.05%, L-asparagine-0.5%, MgSO 4 .7H 2 O-2ml (1 M), CaCl 2 .2H 2 O-1ml, (0.1 M), glucose-5%, agar-2%, and pH-7) was incorporated with a pH indicator (0.007 % Bromo Thymol Blue) [26].…”

Section: Microbialmentioning

confidence: 99%

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Submerged fermentation, purification, and characterization of L-asparaginase from Streptomyces sp. isolated from soil

2018

J App Biol Biotech

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Actinomycetes are most valuable microbes because they are reported to produce different kinds of novel antibiotics and bioactive compounds. L-asparaginase enzyme is a chemotherapeutic agent used in the treatment of cancer. The present study reports production and characterization of purified L-asparaginase from an actinomycetes isolate. The isolate (A-164) was found to be a potent producer of L-asparaginase with an enzyme activity of 26.67 international unit (IU)/ml. Biochemical characterization of the isolate indicated that it belongs to genus Streptomyces. Laboratory scale enzyme production studies were undertaken using the standard medium. Further, the enzyme was purified by salt precipitation, anion exchange chromatography, and size exclusion chromatography. The purified enzyme exhibited the specific activity of 390 IU/mg with 60-fold purification and yield of 42% with a molecular weight of 34 KDa. The purified enzyme demonstrated the highest activity at pH 6.0 and temperature 35°C. The enzyme retained its activity till temperature of 50°C, indicating that it is a thermostable enzyme. A study on the effect of various additives on enzyme activity was also undertaken. The K m and V max values for the purified L-asparaginase were 0.065 mM and 20.80 IU/ml, respectively. The enzyme can further be used for health care and industrial applications.

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Section: Isolation and Characterization Of Actinomycetes And Screeninmentioning

confidence: 98%

Section: Microbialmentioning

confidence: 99%

Submerged fermentation, purification, and characterization of L-asparaginase from Streptomyces sp. isolated from soil

2018

J App Biol Biotech

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“…Twelve strains were inoculated in Czapek-Dox agar plates formulated as modified from Gulati et al (1997) and Mahajan et al (2013)…”

Section: Screening Of L-asparaginase Producersmentioning

confidence: 99%

“…A variety of organisms, from bacteria to mammals, were reported to produce L-asparaginase (Peterson & Ciegler 1969;Mahajan, Saran, Saxena, & Srivastava, 2013;Zuo et al, 2015). The L-asparaginase in current trademarks, like Paronal (Tong et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

<b>Fungal production of the anti-leukemic enzyme L-asparaginase: from screening to medium development

Gonçalves

Maia

Rueda

et al. 2016

Acta Sci. Biol. Sci.

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ABSTRACT. 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.Keywords: therapeutic enzymes, filamentous fungi, acute lymphoblastic leukemia, Penicillium sp., Fusarium sp.Produção fúngica da enzima antileucêmica L-asparaginase: da triagem ao desenvolvimento de um meio de cultivo RESUMO. 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. (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.Palavras-chave: enzimas terapêuticas, fungos filamentosos, leucemia linfocítica aguda, Penicillium sp., Fusarium sp.

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“…Além disso, outro problema envolvido com a enzima bacteriana, é a produção intracelular da mesma. Esse fato torna o processo de extração e purificação da enzima muito difícil e bastante oneroso (MAHAJAN et al, 2013). Diante disso, a busca por outras fontes de L-Asparaginase tem sido alvo de muitas pesquisas, com o objetivo de se obter a produção de enzimas extracelulares pelos micro-organismos, com altos rendimentos, além de apresentar menos efeitos adversos .…”

Section: Leucemia Linfóide Agudaunclassified

Avaliação da produção de L- Asparaginase por fungos isolados do bioma Cerrado

Almeida¹

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A Deus, por mais essa etapa vencida em minha vida.A Professora Dra. Pérola de Oliveira Magalhães Dias Batista, pela oportunidade, confiança e amizade. Seus estímulos, apoio e questionamentos foram fundamentais para a conclusão desse trabalho. Muito obrigada.Ao meu maior incentivador, companheiro de todas as horas, meu amor Bruno.Obrigada pelo carinho, compreensão, apoio e paciência neste período e em todos os momentos da minha vida. Amo muito você.Aos meus pais, Renato e Magda, e ao meu irmão, Mateus, que, mesmo distantes, incentivam e compartilham os grandes momentos da minha vida. Sem vocês, nada disso seria possível.A toda minha família que sempre torceu muito pelas minhas conquistas, especialmente a minha tão amada avó, Maria, que sempre me protege e abençoa em suas orações.As professoras Dra. Maria de Fátima Borin e Dra. Eliete Neves da Silva Guerra, por terem aceitado o convite e participarem como membros da banca de avaliação deste trabalho. Meus sinceros agradecimentos pela atenção dispensada. Além disso, meu muito obrigada pelas palavras de apoio e incentivo nos momentos mais difíceis, serei eternamente grata por tudo.À CAPES pelo suporte financeiro concedido.A todas as pessoas que direta ou indiretamente contribuíram para o desenvolvimento e conclusão deste trabalho, meu muito obrigada. Asparaginase strain producer, were used. Subsequently, 42 fungal strains from the brazilian savanna were isolated and a screening to assess the ability of the same for the production of L-Asparaginase was performed through the halo test. Out of the 42 isolated stains, 22 species showed red halo, which may indicate the production of LAsparaginase. These 22 species were cultivated in the liquid medium under controlled agitation and temperature and only 10 were positive for enzyme production. Then, the top 3 producing strains were grown in different culture conditions to improve the production of L-Asparaginase. Regarding the carbon sources, glucose utilization was essential for enzyme production. L-Proline seems to be the most effective source of nitrogen for producing L-Asparaginase. In addition, were found different optimum pH values of the different fungal strains cultivation.

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A rapid, efficient and sensitive plate assay for detection and screening ofl-asparaginase-producing microorganisms

Cited by 58 publications

References 9 publications

Submerged fermentation, purification, and characterization of L-asparaginase from Streptomyces sp. isolated from soil

Submerged fermentation, purification, and characterization of L-asparaginase from Streptomyces sp. isolated from soil

<b>Fungal production of the anti-leukemic enzyme L-asparaginase: from screening to medium development

Avaliação da produção de L- Asparaginase por fungos isolados do bioma Cerrado

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