{"title":"Supported ionic liquids to purify microbial L-Asparaginase","authors":"","doi":"10.1016/j.bej.2024.109445","DOIUrl":null,"url":null,"abstract":"<div><p>L-Asparaginase (ASNase) is a versatile enzyme that converts L-asparagine into ammonia and aspartic acid. This enzyme has applications in the food industry and health sector. However, high purity ASNase is required, resulting in high production costs. Therefore, in this work, two supported ionic liquids (SILs), specifically silica modified with dimethylbutylpropylammonium chloride ([Si][N<sub>3114</sub>]Cl) or triethylpropylammonium chloride ([Si][N<sub>3222</sub>]Cl), were investigated as alternative adsorption materials to purify ASNase. Different conditions were evaluated to improve enzyme purity, including total protein content in the cell extract, contact time, and SIL/cell extract ratio (w/v). Under optimal conditions using [Si][N<sub>3114</sub>]Cl, a maximum ASNase purification of 6.1-fold is achieved in a single step, resulting from the preferential attachment of other proteins on [Si][N<sub>3114</sub>]Cl SIL. According to the results, hydrophobic interactions rule the selective adsorption of protein impurities from the cell extract by the SIL, thereby increasing the ASNase purification levels. This approach offers a significant advantage, not requiring the desorption and elution of the target enzyme, while envisioning the application of SILs in a flow-through elution approach. The protonation state of protein surface was calculated by computational analysis, revealing that positively charged amino acids such as arginine and lysine block the effective binding of the enzyme to the SILs. Overall, if properly designed, SILs are promising alternative supports for the downstream processing of ASNase from cell extracts.</p></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":null,"pages":null},"PeriodicalIF":3.7000,"publicationDate":"2024-08-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1369703X24002328/pdfft?md5=ad85fe365ade8e6c8077cc484db45acc&pid=1-s2.0-S1369703X24002328-main.pdf","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biochemical Engineering Journal","FirstCategoryId":"5","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1369703X24002328","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
L-Asparaginase (ASNase) is a versatile enzyme that converts L-asparagine into ammonia and aspartic acid. This enzyme has applications in the food industry and health sector. However, high purity ASNase is required, resulting in high production costs. Therefore, in this work, two supported ionic liquids (SILs), specifically silica modified with dimethylbutylpropylammonium chloride ([Si][N3114]Cl) or triethylpropylammonium chloride ([Si][N3222]Cl), were investigated as alternative adsorption materials to purify ASNase. Different conditions were evaluated to improve enzyme purity, including total protein content in the cell extract, contact time, and SIL/cell extract ratio (w/v). Under optimal conditions using [Si][N3114]Cl, a maximum ASNase purification of 6.1-fold is achieved in a single step, resulting from the preferential attachment of other proteins on [Si][N3114]Cl SIL. According to the results, hydrophobic interactions rule the selective adsorption of protein impurities from the cell extract by the SIL, thereby increasing the ASNase purification levels. This approach offers a significant advantage, not requiring the desorption and elution of the target enzyme, while envisioning the application of SILs in a flow-through elution approach. The protonation state of protein surface was calculated by computational analysis, revealing that positively charged amino acids such as arginine and lysine block the effective binding of the enzyme to the SILs. Overall, if properly designed, SILs are promising alternative supports for the downstream processing of ASNase from cell extracts.
期刊介绍:
The Biochemical Engineering Journal aims to promote progress in the crucial chemical engineering aspects of the development of biological processes associated with everything from raw materials preparation to product recovery relevant to industries as diverse as medical/healthcare, industrial biotechnology, and environmental biotechnology.
The Journal welcomes full length original research papers, short communications, and review papers* in the following research fields:
Biocatalysis (enzyme or microbial) and biotransformations, including immobilized biocatalyst preparation and kinetics
Biosensors and Biodevices including biofabrication and novel fuel cell development
Bioseparations including scale-up and protein refolding/renaturation
Environmental Bioengineering including bioconversion, bioremediation, and microbial fuel cells
Bioreactor Systems including characterization, optimization and scale-up
Bioresources and Biorefinery Engineering including biomass conversion, biofuels, bioenergy, and optimization
Industrial Biotechnology including specialty chemicals, platform chemicals and neutraceuticals
Biomaterials and Tissue Engineering including bioartificial organs, cell encapsulation, and controlled release
Cell Culture Engineering (plant, animal or insect cells) including viral vectors, monoclonal antibodies, recombinant proteins, vaccines, and secondary metabolites
Cell Therapies and Stem Cells including pluripotent, mesenchymal and hematopoietic stem cells; immunotherapies; tissue-specific differentiation; and cryopreservation
Metabolic Engineering, Systems and Synthetic Biology including OMICS, bioinformatics, in silico biology, and metabolic flux analysis
Protein Engineering including enzyme engineering and directed evolution.