Continuous refolding of L-asparaginase inclusion bodies using periodic counter-current chromatography

Rajendran, Vivek; Pushpavanam, S.; Jayaraman, Guhan

doi:10.1016/j.chroma.2021.462746

Cited by 8 publications

(7 citation statements)

References 40 publications

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“…The in vitro refolding of insoluble WT‐LAO, single mutant, and 6‐point mutant was performed using the pulse dilution method [22] . This method involves the dropwise addition of the solubilized LAO into the refolding buffer at a flow rate of approximately 0.1 ml/min, thus allowing the addition of a concentrated amount of solubilized protein and the obtention of higher titers of refolded protein than the conventional dilution process [23] . Subsequently, the activity of the refolded LAO was measured.…”

Section: Resultsmentioning

confidence: 99%

“…[22] This method involves the dropwise addition of the solubilized LAO into the refolding buffer at a flow rate of approximately 0.1 ml/min, thus allowing the addition of a concentrated amount of solubilized protein and the obtention of higher titers of refolded protein than the conventional dilution process. [23] Subsequently, the activity of the refolded LAO was measured. An improvement in refolding ability was observed in mutant enzymes, except T204 A.…”

Section: Construction and Characterization Of Duplicate Mutant Enzymesmentioning

confidence: 99%

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Improvement of Heterologous Soluble Expression of L‐amino Acid Oxidase Using Logistic Regression

Nakahara,

Su,

Wakayama

et al. 2024

ChemBioChem

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Successful implementation of enzymes in practical application hinges on the development of efficient mass production techniques. However, in a heterologous expression system, the protein is often unable to fold correctly and, thus, forms inclusion bodies, resulting in the loss of its original activity. In this study, we present a new and more accurate model for predicting amino acids associated with an increased L‐amino acid oxidase (LAO) solubility. Expressing LAO from Rhizoctonia solani in Escherichia coli and combining random mutagenesis and statistical logistic regression, we modified 108 amino acid residues by substituting hydrophobic amino acids with serine and hydrophilic amino acids with alanine. Our results indicated that specific mutations in Euclidean distance, glycine, methionine, and secondary structure increased LAO expression. Furthermore, repeated mutations were performed for LAO based on logistic regression models. The mutated LAO displayed a significantly increased solubility, with the 6‐point and 58‐point mutants showing a 2.64‐ and 4.22‐fold increase, respectively, compared with WT‐LAO. Ultimately, using recombinant LAO in the biotransformation of α‐keto acids indicates its great potential as a biocatalyst in industrial production.

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Section: Resultsmentioning

confidence: 99%

Section: Construction and Characterization Of Duplicate Mutant Enzymesmentioning

confidence: 99%

Improvement of Heterologous Soluble Expression of L‐amino Acid Oxidase Using Logistic Regression

Nakahara,

Su,

Wakayama

et al. 2024

ChemBioChem

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“…Considerable effort has gone into downstream processing involving isolation, solubilization, renaturation (refolding), and purification to obtain the soluble, bioactive protein ( Kante et al, 2018 ). Researchers have achieved up to 50% recovery of functional L-ASNase using various strategies such as the use of strong chaotropic agents ( Kante et al, 2018 ), pulse dilution method ( Upadhyay et al, 2014 ), Freeze-Thaw method ( Singhvi et al, 2021 ), refolding in periodic counter-current chromatography (PCC) ( Rajendran et al, 2022 ), among others. However, these steps are time consuming; require major equipment such as new generation chromatographs, ultrafiltration and diafiltration systems, hydraulic intensifier systems, etc; a large number of reagents such as chaotropic agents, micelles, liposomes, detergents, etc; and use large volumes (generally 1–10 L for mg quantities of protein) ( Clark, 2001 ; Singh et al, 2015 ; Yuan et al, 2015 ).…”

Section: Improvement In Systems For L-asnase Heterologous Expressionmentioning

confidence: 99%

Current state of molecular and metabolic strategies for the improvement of L-asparaginase expression in heterologous systems

et al. 2023

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Heterologous expression of L-asparaginase (L-ASNase) has become an important area of research due to its clinical and food industry applications. This review provides a comprehensive overview of the molecular and metabolic strategies that can be used to optimize the expression of L-ASNase in heterologous systems. This article describes various approaches that have been employed to increase enzyme production, including the use of molecular tools, strain engineering, and in silico optimization. The review article highlights the critical role that rational design plays in achieving successful heterologous expression and underscores the challenges of large-scale production of L-ASNase, such as inadequate protein folding and the metabolic burden on host cells. Improved gene expression is shown to be achievable through the optimization of codon usage, synthetic promoters, transcription and translation regulation, and host strain improvement, among others. Additionally, this review provides a deep understanding of the enzymatic properties of L-ASNase and how this knowledge has been employed to enhance its properties and production. Finally, future trends in L-ASNase production, including the integration of CRISPR and machine learning tools are discussed. This work serves as a valuable resource for researchers looking to design effective heterologous expression systems for L-ASNase production as well as for enzymes production in general.

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“…[ 14 ] Another recent report showed successful on‐column refolding of L‐asparaginase using periodic counter‐current (PCC) chromatography after solubilization. [ 17 ]…”

Section: Introductionmentioning

confidence: 99%

“…[14] Another recent report showed successful on-column refolding of L-asparaginase using periodic counter-current (PCC) chromatography after solubilization. [17] Post-solubilization, the L-asparaginase present as inactive monomers refolds in the active tetrameric form under various physiological conditions and nutritional additives. In this study, the impact of urea concentration, IBs concentration, incubation time, refolding method, pH of refolding buffer, and dilution factor of solubilized IBs (SIBs) in refolding buffer on refolding yield and process intermediate stability has been investigated.…”

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confidence: 99%

Post‐refolding stability considerations for optimization of in‐vitro refolding: L‐asparaginase as a case study

Mihooliya

Nitika

Bhambure

et al. 2022

Biotechnology Journal

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Background L‐Asparaginase is an essential enzyme for the food and biopharmaceutical industry. The stability, however, of L‐asparaginase is widely known to be an issue. Commercial manufacturing of any biopharmaceutical involves hold‐ups during processing, and can result in product loss if stability is an issue, as is the case with L‐asparaginase. This interplay of product intermediate stability and process design is the focus of this investigation. Methods and Results In this study, we propose a strategy to simultaneously increase the refolding yield and stability of refolded L‐asparaginase so as to improve overall process yield. Using one variable at a time (OVAT) experiments, urea (6 M), solubilized inclusion bodies (15 mg/ml), refolding method (step dilution), and pH (8.6) were identified as significant process parameters. A design of experiment (DOE)‐based optimization was then performed for the refolding step. The net outcome was more than a three‐fold increase in enzyme recovery (i.e., 4.90 IU/ml) compared to unoptimized conditions (i.e., 1.26 IU/ml). Further, the L‐asparaginase process intermediate was found to be stable for more than a week at room temperature and 2–8°C, while the unoptimized sample was stable at 2–8°C but did not show any activity at room temperature after 72 h. Conclusions The current study elucidates how process intermediate stability needs to be given due consideration during process optimization, particularly for products such as L‐asparaginase which are labile.

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Continuous refolding of L-asparaginase inclusion bodies using periodic counter-current chromatography

Cited by 8 publications

References 40 publications

Improvement of Heterologous Soluble Expression of L‐amino Acid Oxidase Using Logistic Regression

Improvement of Heterologous Soluble Expression of L‐amino Acid Oxidase Using Logistic Regression

Current state of molecular and metabolic strategies for the improvement of L-asparaginase expression in heterologous systems

Post‐refolding stability considerations for optimization of in‐vitro refolding: L‐asparaginase as a case study

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