{"title":"纯化微生物 L-天冬酰胺酶的支撑离子液体","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":"{\"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. 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引用次数: 0
摘要
左旋天冬酰胺酶(ASNase)是一种多功能酶,可将左旋天冬酰胺转化为氨和天冬氨酸。这种酶可应用于食品工业和卫生部门。然而,ASNase 需要高纯度,因此生产成本较高。因此,在这项工作中,研究了两种支撑离子液体(SIL),特别是用二甲基丁基丙基氯化铵([Si][N]Cl)或三乙基丙基氯化铵([Si][N]Cl)修饰的二氧化硅,作为纯化 ASNase 的替代吸附材料。评估了提高酶纯度的不同条件,包括细胞提取物中的总蛋白含量、接触时间和 SIL/细胞提取物比率(w/v)。在使用[Si][N]Cl的最佳条件下,由于其他蛋白质优先附着在[Si][N]Cl SIL上,ASNase的单步纯化率最高可达6.1倍。结果表明,疏水相互作用使 SIL 能够选择性地吸附细胞提取物中的蛋白质杂质,从而提高了 ASNase 的纯化水平。这种方法具有明显的优势,不需要对目标酶进行解吸和洗脱,同时还能将 SIL 应用于流动洗脱方法。通过计算分析蛋白质表面的质子化状态,发现带正电荷的氨基酸(如精氨酸和赖氨酸)会阻碍酶与 SILs 的有效结合。总之,如果设计得当,SILs 将成为从细胞提取物中提取 ASNase 的下游处理过程中很有前景的替代支持物。
Supported ionic liquids to purify microbial L-Asparaginase
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.