Improvement of the activity of l-asparaginase I improvement of the catalytic activity of l-asparaginase I from Bacillus megaterium H-1 by in vitro directed evolution

Lu, Xiao‐Hong; Chen, Juhua; Jiao, Linshu; Zhong, Lei; Lu, Zhaoxin; Zhang, Chong; Lü, Fang

doi:10.1016/j.jbiosc.2019.06.001

Cited by 17 publications

(9 citation statements)

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“…However, their administration is not free from severe side effects. A number of experiments have been undertaken to improve the properties of therapeutic lasparaginases (Nguyen et al, 2016;Lopes et al, 2017;Aghaeepoor et al, 2018;Maggi et al, 2017;Verma et al, 2014;Lu et al, 2019;Kotzia & Labrou, 2009;Rigouin et al, 2017). However, none of the improved variants could be introduced into clinical practice.…”

Section: Introductionmentioning

confidence: 99%

Structural and biophysical studies of new L-asparaginase variants: lessons from random mutagenesis of the prototypic Escherichia coli Ntn-amidohydrolase

Loch

Klonecka

Kądziołka

et al. 2022

Acta Cryst Sect D Struct Biol

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This work reports the results of random mutagenesis of the Escherichia coli class 2 L-asparaginase EcAIII belonging to the Ntn-hydrolase family. New variants of EcAIII were studied using structural, biophysical and bioinformatic methods. Activity tests revealed that the L-asparaginase activity is abolished in all analyzed mutants with the absence of Arg207, but some of them retained the ability to undergo the autoproteolytic maturation process. The results of spectroscopic studies and the determined crystal structures showed that the EcAIII fold is flexible enough to accept different types of mutations; however, these mutations may have a diverse impact on the thermal stability of the protein. The conclusions from the experiments are grouped into six lessons focused on (i) the adaptation of the EcAIII fold to new substitutions, (ii) the role of Arg207 in EcAIII activity, (iii) a network of residues necessary for autoprocessing, (iv) the complexity of the autoprocessing reaction, (v) the conformational changes observed in enzymatically inactive variants and (vi) the cooperativity of the EcAIII dimer subunits. Additionally, the structural requirements (pre-maturation checkpoints) that are necessary for the initiation of the autocleavage of Ntn-hydrolases have been classified. The findings reported in this work provide useful hints that should be considered before planning enzyme-engineering experiments aimed at the design of proteins for therapeutic applications. This is especially important for L-asparaginases that can be utilized in leukemia therapy, as alternative therapeutics are urgently needed to circumvent the severe side effects associated with the currently used enzymes.

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Section: Introductionmentioning

confidence: 99%

Structural and biophysical studies of new L-asparaginase variants: lessons from random mutagenesis of the prototypic Escherichia coli Ntn-amidohydrolase

Loch

Klonecka

Kądziołka

et al. 2022

Acta Cryst Sect D Struct Biol

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“…Error-prone polymerase chain reaction (PCR) and DNA shuffling Reduction of hydrogen bonds, and incorporation of flexible residues at an active site Thr15 ~21 fold improvement in catalytic activity, tolerance wider pH range and temperatures (Lu et al, 2019) 57 Fig. 1a Fig.…”

Section: Bacillus Amyloliquefaciens Ss35 Uv Irradiationmentioning

confidence: 99%

Molecular biology interventions for activity improvement and production of industrial enzymes

Bhatia

Narisetty

Kumar

et al. 2021

Bioresource Technology

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“…coli BL21 (DE3) harboring plasmid containing the GmASNase gene was inoculated at 37 • C and an agitation rate of 180 rpm until an OD 600 of 0.6-0.8 was reached. Protein expression was induced by adding IPTG at a final concentration of 100 mg mL −1 , followed by incubation at 16 • C for 20 h [10,28]. The cells were collected by centrifugation at 8000× g for 5 min, before being suspended with Tris-HCl buffer (20 mM Tris-HCL, 300 mM NaCl, pH 8.0).…”

Section: Expression and Purification Of Mycobacterium Gordonae L-asparaginase In Escherichia Colimentioning

confidence: 99%

“…The recombinant GmASNase (3 µg/µL) was added into 4× protein loading buffer solution, and was then placed in a boiling water bath for 3-5 min. The bands were stained with Coomassie Brilliant Blue R250 and decolorized with a mixed aqueous solution of 10% ethanol and 10% acetic acid for easy observation [10,28].…”

Section: Expression and Purification Of Mycobacterium Gordonae L-asparaginase In Escherichia Colimentioning

confidence: 99%

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Characterization of a Novel L-Asparaginase from Mycobacterium gordonae with Acrylamide Mitigation Potential

Chi

Chen

Jiao

et al. 2021

Foods

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L-asparaginase (E.C.3.5.1.1) is a well-known agent that prevents the formation of acrylamide both in the food industry and against childhood acute lymphoblastic leukemia in clinical settings. The disadvantages of L-asparaginase, which restrict its industrial application, include its narrow range of pH stability and low thermostability. In this study, a novel L-asparaginase from Mycobacterium gordonae (GmASNase) was cloned and expressed in Escherichia coli BL21 (DE3). GmASNase was found to be a tetramer with a monomeric size of 32 kDa, sharing only 32% structural identity with Helicobacter pylori L-asparaginases in the Protein Data Bank database. The purified GmASNase had the highest specific activity of 486.65 IU mg−1 at pH 9.0 and 50 °C. In addition, GmASNase possessed superior properties in terms of stability at a wide pH range of 5.0–11.0 and activity at temperatures below 40 °C. Moreover, GmASNase displayed high substrate specificity towards L-asparagine with Km, kcat, and kcat/Km values of 6.025 mM, 11,864.71 min−1 and 1969.25 mM−1min−1, respectively. To evaluate its ability to mitigate acrylamide, GmASNase was used to treat potato chips prior to frying, where the acrylamide content decreased by 65.09% compared with the untreated control. These results suggest that GmASNase is a potential candidate for applications in the food industry.

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Improvement of the activity of l-asparaginase I improvement of the catalytic activity of l-asparaginase I from Bacillus megaterium H-1 by in vitro directed evolution

Cited by 17 publications

References 50 publications

Structural and biophysical studies of new L-asparaginase variants: lessons from random mutagenesis of the prototypic Escherichia coli Ntn-amidohydrolase

Structural and biophysical studies of new L-asparaginase variants: lessons from random mutagenesis of the prototypic Escherichia coli Ntn-amidohydrolase

Molecular biology interventions for activity improvement and production of industrial enzymes

Characterization of a Novel L-Asparaginase from Mycobacterium gordonae with Acrylamide Mitigation Potential

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