Effect of osmolytes on the activity of anti-cancer enzyme L-Asparaginase II from Erwinia chrysanthemi

Wlodarczyk, Samarina Rodrigues; Costa-Silva, Tales A.; Pessoa, Adalberto; Madeira, Pedro P.; Monteiro, Gisele

doi:10.1016/j.procbio.2019.03.009

Cited by 20 publications

(6 citation statements)

References 49 publications

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“…As tagged proteins are normally used in single‐step affinity purification and automated CFPS processes, 26‐28 an L‐ASNase enzyme with a His‐tag fused at the N‐terminal position – named L‐ASNase (I) – was expressed. In fact, a previous study has shown no significant loss of L‐ASNase activity when the recombinant protein was fused to a His‐tag at the N‐terminus 29 . Other L‐ASNase genetic constructs were also tested for expression in this CFPS system: (II) L‐ASNase with no His‐tag, (III) L‐ASNase with a His‐tag at the C‐terminus, and an (IV) L‐ASNase with a His‐tag at the N‐terminus, which contained a different protease cleavage site between the His‐tag and the L‐ASNase protein sequence in comparison to L‐ASNase (I) (Fig.…”

Section: Resultsmentioning

confidence: 95%

Development of a cell‐free protein synthesis protocol to rapidly screenL‐asparaginaseproteoforms by enzymatic activity

Lima

Menezes

Costa

et al. 2021

J of Chemical Tech & Biotech

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BACKGROUND Cell‐free protein synthesis (CFPS) technology has emerged as a powerful tool for a variety of biotechnological applications, including the expression of different classes of biopharmaceutical products. L‐Asparaginase (E.C. Number: 3.5.1.1, L‐asparagine amidohydrolase) (L‐ASNase) is an important biopharmaceutical used to treat leukemia, but expression of multiple proteoforms in CFPS systems and rapid characterization using standard colorimetric methods has not yet been fully exploited. Herein, recombinant expression and characterization of an L‐ASNase from Erwinia chrysanthemi (Erwinase) using a new CFPS protocol is reported. RESULTS Expression and quantification of the enzymatic activity of a soluble his‐tagged L‐ASNase directly from a CFPS reaction was successfully achieved. Purification of the protein was not required in order to assess its biological activity. Activity of L‐ASNase was significantly higher than the control reaction (7.07 ± 0.68 U mL–1 vs. 1.83 ± 0.14 U mL–1, respectively). Expression of a mutant Erwinase proteoform – V293M – was also achieved and it presented a similar enzymatic activity. No significant loss in L‐ASNase enzymatic activity was noticed after removal of cyclic AMP, spermidine, transfer RNA, T7 RNA polymerase and, especially, ammonium acetate (a common interference in ASNase enzymatic assays) from the CFPS reaction. CONCLUSION The protocol developed in this work will facilitate the screening of novel clinically‐relevant L‐ASNase proteoforms. © 2021 Society of Chemical Industry (SCI).

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Section: Resultsmentioning

confidence: 95%

Development of a cell‐free protein synthesis protocol to rapidly screenL‐asparaginaseproteoforms by enzymatic activity

Lima

Menezes

Costa

et al. 2021

J of Chemical Tech & Biotech

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“…4 b and c). Therefore, the protein disaggregation can be explained by the favorite hydration of the enzyme after mixing with osmolyte, which produces intra-chain bonds that can favor equilibrium in the direction of active conformations [ 50 ].…”

Section: Resultsmentioning

confidence: 99%

Stable and effective eco-enzyme cocktails in powder and liquid form of Stachybotrys microspora used as detergent additives

Ben Hmad,

Gargouri

2024

Heliyon

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“…helps stabilizing enzymes [29][30][31][32]. Protective effect of polyols was reported in many recent studies in different experimental situations and exposition to stresses (high pressure or high temperature, presence of non-aqueous solvents) [33][34][35][36][37].…”

Section: Chemical Stabilizer Additionmentioning

confidence: 89%

From Enzyme Stability to Enzymatic Bioelectrode Stabilization Processes

et al. 2021

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Bioelectrocatalysis using redox enzymes appears as a sustainable way for biosensing, electricity production, or biosynthesis of fine products. Despite advances in the knowledge of parameters that drive the efficiency of enzymatic electrocatalysis, the weak stability of bioelectrodes prevents large scale development of bioelectrocatalysis. In this review, starting from the understanding of the parameters that drive protein instability, we will discuss the main strategies available to improve all enzyme stability, including use of chemicals, protein engineering and immobilization. Considering in a second step the additional requirements for use of redox enzymes, we will evaluate how far these general strategies can be applied to bioelectrocatalysis.

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Effect of osmolytes on the activity of anti-cancer enzyme L-Asparaginase II from Erwinia chrysanthemi

Cited by 20 publications

References 49 publications

Development of a cell‐free protein synthesis protocol to rapidly screenL‐asparaginaseproteoforms by enzymatic activity

Development of a cell‐free protein synthesis protocol to rapidly screenL‐asparaginaseproteoforms by enzymatic activity

Stable and effective eco-enzyme cocktails in powder and liquid form of Stachybotrys microspora used as detergent additives

From Enzyme Stability to Enzymatic Bioelectrode Stabilization Processes

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