Recombinant Erwinia carotovora l-asparaginase II production in Escherichia coli fed-batch cultures

Roth, Gustavo; Nunes, J.; Rosado, Leonardo Astolfi; Bizarro, Cristiano Valim; Volpato, Giandra; Nunes, Cláudia Paiva; Renard, Gaby; Basso, Luiz Augusto; Santos, Daniel Silas Veras dos; Chies, Jocelei Maria

doi:10.1590/s0104-66322013000200003

Cited by 27 publications

(22 citation statements)

References 41 publications

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“…The present source of L-asparaginase was pharmaceutically may be a relevant alternative because L-asparaginase with low Km values was preferable for anti-cancer therapy [27] .…”

Section: Resultsmentioning

confidence: 99%

Optimization, purification, and characterization of L-asparaginase fromActinomycetales bacteriumBkSoiiA

Dash

Mohapatra

Maiti

2014

Preparative Biochemistry and Biotechnology

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Actinobacteria are promising source of a wide range of important enzymes, some of which are produced in industrial scale, with others yet to be harnessed. L-Asparaginase is used as an antineoplastic agent. The present work deals with the production and optimization of L-asparaginase from Actinomycetales bacterium BkSoiiA using submerged fermentation in M9 medium. Production optimization resulted in a modified M9 medium with yeast extract and fructose as carbon and nitrogen sources, respectively, at pH 8.0, incubated for 120 hr at 30 ± 2 °C. The crude enzyme was purified to near homogeneity by ammonium sulfate precipitation following dialysis, ion-exchange column chromatography, and finally gel filtration. The sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) revealed an apparent molecular weight of 57 kD. The enzyme was purified 95.06-fold and showed a final specific activity of 204.37 U/mg with 3.49% yield. The purified enzyme showed maximum activity at a pH 10.0 and was stable at pH 7.0 to 9.0. The enzyme was activated by Mn(2+) and strongly inhibited by Ba(2+). All these preliminary characterization suggests that the L-asparaginase from the source may be a tool useful to pharmaceutical industries after further research.

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“…The present source of L-asparaginase was pharmaceutically may be a relevant alternative because L-asparaginase with low Km values was preferable for anti-cancer therapy [27] .…”

Section: Resultsmentioning

confidence: 99%

Optimization, purification, and characterization of L-asparaginase fromActinomycetales bacteriumBkSoiiA

Dash

Mohapatra

Maiti

2014

Preparative Biochemistry and Biotechnology

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“…The combination of recombinant DNA technology and HDC in the fed-batch process, allows enzyme production in much larger quantities than those obtained in traditional processes (Nakagawa et al, 1995; Roth et al, 2013) and enabled the reduction of the culture volume from 20,000 to 300 L in the Medac's Spectrila® production process (European Medicines Agency, 2015; Wacker Biotech, 2016). The application of this technology to the production of ASNase with the optimization of the cultivation process and an effective purification strategy increased the productivity, the yield and the quality of the ASNase produced, reducing the overall production costs while contributing to the technical-economic feasibility of the process.…”

Section: L-asparaginase Manufacturing: Production Process and Purificmentioning

confidence: 99%

Development of L-Asparaginase Biobetters: Current Research Status and Review of the Desirable Quality Profiles

Brumano

Silva

Costa-Silva

et al. 2019

Front. Bioeng. Biotechnol.

144

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L-Asparaginase (ASNase) is a vital component of the first line treatment of acute lymphoblastic leukemia (ALL), an aggressive type of blood cancer expected to afflict over 53,000 people worldwide by 2020. More recently, ASNase has also been shown to have potential for preventing metastasis from solid tumors. The ASNase treatment is, however, characterized by a plethora of potential side effects, ranging from immune reactions to severe toxicity. Consequently, in accordance with Quality-by-Design (QbD) principles, ingenious new products tailored to minimize adverse reactions while increasing patient survival have been devised. In the following pages, the reader is invited for a brief discussion on the most recent developments in this field. Firstly, the review presents an outline of the recent improvements on the manufacturing and formulation processes, which can severely influence important aspects of the product quality profile, such as contamination, aggregation and enzymatic activity. Following, the most recent advances in protein engineering applied to the development of biobetter ASNases (i.e., with reduced glutaminase activity, proteolysis resistant and less immunogenic) using techniques such as site-directed mutagenesis, molecular dynamics, PEGylation, PASylation and bioconjugation are discussed. Afterwards, the attention is shifted toward nanomedicine including technologies such as encapsulation and immobilization, which aim at improving ASNase pharmacokinetics. Besides discussing the results of the most innovative and representative academic research, the review provides an overview of the products already available on the market or in the latest stages of development. With this, the review is intended to provide a solid background for the current product development and underpin the discussions on the target quality profile of future ASNase-based pharmaceuticals.

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“…The K m value of rBliAIII is 0.67×10 −3 M which showed that it has a good substrate affinity toward Lasparagine. rBliAIII has a good affinity toward the substrate when compared to various [42] Kinetic parameters like K m , k cat , and k cat /K m of E. coli, Erwinia spp., and B. licheniformis (present study) Lasparaginase have been compared microbial asparaginases. rBliAIII can serve as a potential substitute for the commercially available asparaginase sources.…”

Section: Resultsmentioning

confidence: 99%

Characterization of a Recombinant Glutaminase-Free l-Asparaginase (ansA3) Enzyme with High Catalytic Activity from Bacillus licheniformis

Sudhir

Dave

Prajapati

et al. 2014

Appl Biochem Biotechnol

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L-Asparaginase (3.5.1.1) is an enzyme widely used to treat the acute lymphoblastic leukemia. Two genes coding for L-asparaginase (ansA1 and ansA3) from Bacillus licheniformis MTCC 429 were cloned and overexpressed in Escherichia coli BL21 (DE3) cells. The recombinant proteins were purified to homogeneity by one-step purification process and further characterized for various biochemical parameters. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that both the enzymes are monomers of ∼37 kDa. Recombinant ansA1 was found to be highly unstable, and recombinant ansA3 was catalytically active and stable, which showed an optimum activity of 407.65 IU/mg at 37 °C and pH 8. Recombinant ansA3 showed higher substrate specificity for L-asparagine with negligible glutaminase activity. Kinetic parameters like K m , V max, k cat, and k cat/K m were calculated for recombinant ansA3.

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Recombinant Erwinia carotovora l-asparaginase II production in Escherichia coli fed-batch cultures

Cited by 27 publications

References 41 publications

Optimization, purification, and characterization of L-asparaginase fromActinomycetales bacteriumBkSoiiA

Optimization, purification, and characterization of L-asparaginase fromActinomycetales bacteriumBkSoiiA

Development of L-Asparaginase Biobetters: Current Research Status and Review of the Desirable Quality Profiles

Characterization of a Recombinant Glutaminase-Free l-Asparaginase (ansA3) Enzyme with High Catalytic Activity from Bacillus licheniformis

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