Journal of separation science最新文献

{"title":"Affinity Chromatography With Selective Nucleic Acid Hybridization: Direct Coupling With Size Exclusion Chromatography","authors":"Martin Gilar,&nbsp;Brittney Racioppo,&nbsp;Beatrice Muriithi,&nbsp;Kevin Wyndham","doi":"10.1002/jssc.70555","DOIUrl":"https://doi.org/10.1002/jssc.70555","url":null,"abstract":"<p>Affinity liquid chromatography (aLC) with immobilized oligonucleotides provides high selectivity for nucleic acid targets through on-column hybridization. We describe the preparation and evaluation of prototype 2.1 mm × 20 mm affinity columns packed with a 3.5 µm nonporous polystyrene/divinylbenzene sorbent covalently modified with streptavidin. Two different affinity columns were prepared by immobilizing biotinylated oligonucleotides on-column via biotin-streptavidin bond: (i) biotinylated 25-nt oligodeoxythymidine, dT25 or (ii) biotinylated 21 nt TP53 oligonucleotide. dT25 column was used for poly(A) denylated messenger RNA (mRNA) and adenosine oligonucleotides variants analysis. The TP53 column was applied to single-nucleotide polymorphism analysis. Nucleic acid binding was performed in 0.1 M sodium phosphate buffer (pH 7.4); retained species were eluted with water or 10% acetonitrile solution in water. We demonstrate that dT25 column selectively retains poly(A)-tailed EPO, EGFP, and Cas9 mRNAs, while tailless mRNA is unretained. dT25 column resolved dA14–dA25 oligoadenosine length variants and dA25 variants bearing cytidine substitutions. The TP53 column baseline-separated a fully complementary 21-nt sequence from single-nucleotide mismatches. Direct coupling of the dT25 column with a 250 Å, 1.7 µm size exclusion chromatography (SEC) column enabled improved poly(A) tail sizing after RNase T1 digestion of intact mRNA; mRNA recovery exceeded 98%, and carryover remained below 0.1% after cleanup.</p>","PeriodicalId":17098,"journal":{"name":"Journal of separation science","volume":"49 10","pages":""},"PeriodicalIF":3.6,"publicationDate":"2026-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jssc.70555","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149206053","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The pH Modifier Moonlighting on the Stationary Phase: HFIP's Unrecognized Role in Oligonucleotide IP-RP Selectivity","authors":"Joshua D. Jones,&nbsp;Matthew J. Sorensen,&nbsp;Todd D. Maloney","doi":"10.1002/jssc.70558","DOIUrl":"https://doi.org/10.1002/jssc.70558","url":null,"abstract":"<div>\u0000 \u0000 <p>Ion-pairing reversed phase (IP-RP) liquid chromatography is the predominant separation mode for characterizing synthetic oligonucleotide therapeutics, offering unique selectivity for small chemical impurities that are difficult to resolve by other techniques. Although not fully understood, the generally accepted mechanism driving retention in IP-RP is a positively charged, hydrophobic alkylamine adsorbing on the stationary phase and the negatively charged oligonucleotide is retained via electrostatic interaction. However, this model does not fully account for the well documented differences in retention and selectivity observed when the mobile phase acid modifier is changed from acetic acid to 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), suggesting our current mechanistic understanding is incomplete. Here, we perform a systematic evaluation to elucidate how HFIP contributes to IP-RP chromatography for a fully phosphorothioate (PS) modified antisense oligonucleotide. We identified that HFIP interacts directly with C18 stationary phases and has higher retentivity than dibutylamine, providing the first evidence that a mixed mode pseudo-stationary phase comprising both amine and HFIP may exist. We further demonstrate that the protonation state of HFIP affects both alkylamine retentivity and oligonucleotide impurity selectivity. These findings suggest that the IP-RP separation mechanism is more complex than previously appreciated, with HFIP serving as an active, pH-responsive component of the pseudo-stationary phase rather than a passive pH modifier. This mechanistic insight has practical implications for method development, as mobile phase pH near the <i>p</i>K_a of HFIP becomes a previously unrecognized parameter for tuning oligonucleotide selectivity.</p>\u0000 </div>","PeriodicalId":17098,"journal":{"name":"Journal of separation science","volume":"49 10","pages":""},"PeriodicalIF":3.6,"publicationDate":"2026-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149205981","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The pH Modifier Moonlighting on the Stationary Phase: HFIP's Unrecognized Role in Oligonucleotide IP-RP Selectivity","authors":"Joshua D. Jones,&nbsp;Matthew J. Sorensen,&nbsp;Todd D. Maloney","doi":"10.1002/jssc.70558","DOIUrl":"https://doi.org/10.1002/jssc.70558","url":null,"abstract":"<div>\u0000 \u0000 <p>Ion-pairing reversed phase (IP-RP) liquid chromatography is the predominant separation mode for characterizing synthetic oligonucleotide therapeutics, offering unique selectivity for small chemical impurities that are difficult to resolve by other techniques. Although not fully understood, the generally accepted mechanism driving retention in IP-RP is a positively charged, hydrophobic alkylamine adsorbing on the stationary phase and the negatively charged oligonucleotide is retained via electrostatic interaction. However, this model does not fully account for the well documented differences in retention and selectivity observed when the mobile phase acid modifier is changed from acetic acid to 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), suggesting our current mechanistic understanding is incomplete. Here, we perform a systematic evaluation to elucidate how HFIP contributes to IP-RP chromatography for a fully phosphorothioate (PS) modified antisense oligonucleotide. We identified that HFIP interacts directly with C18 stationary phases and has higher retentivity than dibutylamine, providing the first evidence that a mixed mode pseudo-stationary phase comprising both amine and HFIP may exist. We further demonstrate that the protonation state of HFIP affects both alkylamine retentivity and oligonucleotide impurity selectivity. These findings suggest that the IP-RP separation mechanism is more complex than previously appreciated, with HFIP serving as an active, pH-responsive component of the pseudo-stationary phase rather than a passive pH modifier. This mechanistic insight has practical implications for method development, as mobile phase pH near the <i>p</i>K_a of HFIP becomes a previously unrecognized parameter for tuning oligonucleotide selectivity.</p>\u0000 </div>","PeriodicalId":17098,"journal":{"name":"Journal of separation science","volume":"49 10","pages":""},"PeriodicalIF":3.6,"publicationDate":"2026-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149206212","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Affinity Chromatography With Selective Nucleic Acid Hybridization: Direct Coupling With Size Exclusion Chromatography","authors":"Martin Gilar,&nbsp;Brittney Racioppo,&nbsp;Beatrice Muriithi,&nbsp;Kevin Wyndham","doi":"10.1002/jssc.70555","DOIUrl":"https://doi.org/10.1002/jssc.70555","url":null,"abstract":"<p>Affinity liquid chromatography (aLC) with immobilized oligonucleotides provides high selectivity for nucleic acid targets through on-column hybridization. We describe the preparation and evaluation of prototype 2.1 mm × 20 mm affinity columns packed with a 3.5 µm nonporous polystyrene/divinylbenzene sorbent covalently modified with streptavidin. Two different affinity columns were prepared by immobilizing biotinylated oligonucleotides on-column via biotin-streptavidin bond: (i) biotinylated 25-nt oligodeoxythymidine, dT25 or (ii) biotinylated 21 nt TP53 oligonucleotide. dT25 column was used for poly(A) denylated messenger RNA (mRNA) and adenosine oligonucleotides variants analysis. The TP53 column was applied to single-nucleotide polymorphism analysis. Nucleic acid binding was performed in 0.1 M sodium phosphate buffer (pH 7.4); retained species were eluted with water or 10% acetonitrile solution in water. We demonstrate that dT25 column selectively retains poly(A)-tailed EPO, EGFP, and Cas9 mRNAs, while tailless mRNA is unretained. dT25 column resolved dA14–dA25 oligoadenosine length variants and dA25 variants bearing cytidine substitutions. The TP53 column baseline-separated a fully complementary 21-nt sequence from single-nucleotide mismatches. Direct coupling of the dT25 column with a 250 Å, 1.7 µm size exclusion chromatography (SEC) column enabled improved poly(A) tail sizing after RNase T1 digestion of intact mRNA; mRNA recovery exceeded 98%, and carryover remained below 0.1% after cleanup.</p>","PeriodicalId":17098,"journal":{"name":"Journal of separation science","volume":"49 10","pages":""},"PeriodicalIF":3.6,"publicationDate":"2026-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jssc.70555","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149206213","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Breaking the Decolorization Recovery Trade-off: Hybrid Adsorbent System Coupled With Response Surface Methodology and Artificial Neural Network for Efficient Pullulan Purification","authors":"Qingli Guan,&nbsp;Xinyue Zhang,&nbsp;Wenbin Liu,&nbsp;Tingbin Zhao,&nbsp;Weifeng Cao,&nbsp;Changsheng Qiao","doi":"10.1002/jssc.70557","DOIUrl":"10.1002/jssc.70557","url":null,"abstract":"<div>\u0000 \u0000 <p>To overcome the long-standing “decolorization-recovery trade-off” in pullulan purification, this study innovatively developed a synergistic hybrid decolorization system integrating D392 resin and 325-mesh activated carbon, guided by a hybrid framework combining response surface methodology (RSM) and backpropagation artificial neural network (BP-ANN) for mechanistic exploration and high-precision prediction. Initial single-adsorbent optimization using RSM achieved high decolorization rates of 92.5% (1.8% resin) and 92.0% (1.4% activated carbon), but suffered from poor pullulan recoveries of only 80.14% and 77.14%, respectively. For the first time, mechanistic analysis revealed that pigment adsorption from complex pullulan fermentation broth (mixed pigment fractions) follows a monolayer Langmuir model for D392 resin and a multilayer Freundlich model for activated carbon, both conforming to pseudo-second-order kinetics, providing a theoretical foundation for synergistic system design. Considering the intrinsic limitation of quadratic response surface models for describing highly nonlinear multi-adsorbent interactions, a BP-ANN network was further introduced, and an independent test subset was applied to objectively compare predictive performance. Leveraging these distinct adsorption properties, sequential adsorption was optimized using RSM and BP-ANN, where the ANN demonstrated superior prediction accuracy. Under ANN-optimized conditions (0.17% resin and 0.88% activated carbon), the hybrid system achieved 92% decolorization and 90.4% recovery, drastically reducing resin consumption by 90.76% and activated carbon by 37.14% compared with single-adsorbent processes. Comprehensive characterization (ultraviolet-visible spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction, nuclear magnetic resonance, and scanning electron microscopy) confirmed that the native molecular structure and functional properties of pullulan were fully preserved. This work not only breaks the conventional trade-off between decolorization efficiency and pullulan yield but also provides a novel, cost-effective methodological paradigm for polysaccharide purification.</p>\u0000 </div>","PeriodicalId":17098,"journal":{"name":"Journal of separation science","volume":"49 10","pages":""},"PeriodicalIF":3.6,"publicationDate":"2026-10-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149181498","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Recent Advances in Sample Preparation for Cholesterol and Oxysterol Analysis in Complex Biological Matrices","authors":"Haizhen Zhang,&nbsp;Longchan Liu,&nbsp;Haoyue Zhang,&nbsp;Linnan Li,&nbsp;Zhengtao Wang,&nbsp;Li Yang","doi":"10.1002/jssc.70539","DOIUrl":"https://doi.org/10.1002/jssc.70539","url":null,"abstract":"<div>\u0000 \u0000 <p>Cholesterol and oxysterols are key molecules that regulate physiological processes and participate in the occurrence and progression of various major diseases. Accurate and efficient analytical detection of these substances in complex biological matrices is critical for disease mechanism research and biomarker discovery. However, challenges including complex biological matrices, large differences in analyte abundance, susceptibility to auto‑oxidation, low ionization efficiency, and difficulty in isomer separation pose severe obstacles to reliable analysis, making sample preparation the core step for accurate quantification and valid identification. Continuous innovations have been achieved in sample preparation techniques, such as liquid–liquid extraction, solid‑phase extraction, deep eutectic solvent extraction, molecularly imprinted extraction, derivatization, as well as integrated and automated workflows. These advances improve extraction efficiency, selectivity, antioxidant protection, and throughput, while effectively reducing matrix interference and artificial oxidation. This paper systematically reviews the research progress in sample preparation for cholesterol and oxysterol analysis in complex biological matrices from 2015 to 2026, highlights the technical advantages, application scenarios, and current limitations of various pretreatment strategies, and prospects the development trends toward greening, intelligence, and high throughput.</p>\u0000 </div>","PeriodicalId":17098,"journal":{"name":"Journal of separation science","volume":"49 10","pages":""},"PeriodicalIF":3.6,"publicationDate":"2026-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149146572","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Controlled Silica Hydration Enables Mechanism-guided Green Solid-Phase Extraction for Steryl Glucoside Determination in Biodiesel","authors":"Muhammad Rida'i,&nbsp;Salisa Chumsantea,&nbsp;Sarttrawut Tulaphol,&nbsp;Kornkanok Aryusuk","doi":"10.1002/jssc.70553","DOIUrl":"https://doi.org/10.1002/jssc.70553","url":null,"abstract":"<div>\u0000 \u0000 <p>Steryl glucosides are minor lipid contaminants in biodiesel that can cause fuel filter plugging and adversely affect fuel quality, even at trace concentrations. Reliable determination of steryl glucoside is therefore essential for biodiesel quality control. Solid-phase extraction (SPE) is widely used for sample pretreatment prior to chromatographic analysis; however, conventional silica-based SPE protocols typically require hazardous organic solvents for cartridge preconditioning. In this study, a controlled silica hydration-based SPE method was developed for the pretreatment of steryl glucoside in crude biodiesel prior to high-performance size-exclusion chromatography coupled with evaporative light-scattering detection using a 100-Å Phenogel column. The effect of water volume (0.05–0.50 mL) used for silica preconditioning was systematically investigated. Following SPE, retained steryl glucoside was recovered by heptane washing and elution with acetone/methanol (9:1, v/v). Preconditioning with 0.075 mL of water provided the highest steryl glucoside concentration (106.29 µg/mL), approximately four times the concentration obtained with non-preconditioned silica (27.06 µg/mL) and substantially higher than those obtained with conventional heptane- (53.70 µg/mL) and acetone/methanol-preconditioned (76.73 µg/mL) cartridges. The method exhibited limits of detection and quantification of 8.07 and 24.47 µg/mL, respectively. The enhanced extraction efficiency is attributed to controlled hydration of surface silanol groups, which establishes a balanced physicochemical environment favorable for steryl glucoside adsorption while substantially reducing the consumption of hazardous organic solvents. The proposed controlled silica hydration-based SPE method provides a simple, environmentally friendly, and mechanistically guided pretreatment strategy for the determination of steryl glucoside in biodiesel and may also be applicable to the extraction of other polar lipid analytes.</p>\u0000 </div>","PeriodicalId":17098,"journal":{"name":"Journal of separation science","volume":"49 10","pages":""},"PeriodicalIF":3.6,"publicationDate":"2026-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149146571","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Controlled Silica Hydration Enables Mechanism-guided Green Solid-Phase Extraction for Steryl Glucoside Determination in Biodiesel","authors":"Muhammad Rida'i,&nbsp;Salisa Chumsantea,&nbsp;Sarttrawut Tulaphol,&nbsp;Kornkanok Aryusuk","doi":"10.1002/jssc.70553","DOIUrl":"https://doi.org/10.1002/jssc.70553","url":null,"abstract":"<div>\u0000 \u0000 <p>Steryl glucosides are minor lipid contaminants in biodiesel that can cause fuel filter plugging and adversely affect fuel quality, even at trace concentrations. Reliable determination of steryl glucoside is therefore essential for biodiesel quality control. Solid-phase extraction (SPE) is widely used for sample pretreatment prior to chromatographic analysis; however, conventional silica-based SPE protocols typically require hazardous organic solvents for cartridge preconditioning. In this study, a controlled silica hydration-based SPE method was developed for the pretreatment of steryl glucoside in crude biodiesel prior to high-performance size-exclusion chromatography coupled with evaporative light-scattering detection using a 100-Å Phenogel column. The effect of water volume (0.05–0.50 mL) used for silica preconditioning was systematically investigated. Following SPE, retained steryl glucoside was recovered by heptane washing and elution with acetone/methanol (9:1, v/v). Preconditioning with 0.075 mL of water provided the highest steryl glucoside concentration (106.29 µg/mL), approximately four times the concentration obtained with non-preconditioned silica (27.06 µg/mL) and substantially higher than those obtained with conventional heptane- (53.70 µg/mL) and acetone/methanol-preconditioned (76.73 µg/mL) cartridges. The method exhibited limits of detection and quantification of 8.07 and 24.47 µg/mL, respectively. The enhanced extraction efficiency is attributed to controlled hydration of surface silanol groups, which establishes a balanced physicochemical environment favorable for steryl glucoside adsorption while substantially reducing the consumption of hazardous organic solvents. The proposed controlled silica hydration-based SPE method provides a simple, environmentally friendly, and mechanistically guided pretreatment strategy for the determination of steryl glucoside in biodiesel and may also be applicable to the extraction of other polar lipid analytes.</p>\u0000 </div>","PeriodicalId":17098,"journal":{"name":"Journal of separation science","volume":"49 10","pages":""},"PeriodicalIF":3.6,"publicationDate":"2026-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149146597","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Immobilization of Pepsin on Different Nanomaterials and Its Application in Obtaining Bioactive Peptides","authors":"Annie N. Alves,&nbsp;Márjorie C. P. Porfirio,&nbsp;Priscilla A. Nascimento,&nbsp;Jéssica F. Borges,&nbsp;Wenrong Yang,&nbsp;Motilal Mathesh,&nbsp;Paulo Bonomo,&nbsp;Rafael da C. I. Fontan,&nbsp;Renata C. F. Bonomo","doi":"10.1002/jssc.70546","DOIUrl":"https://doi.org/10.1002/jssc.70546","url":null,"abstract":"<p>Enzyme immobilization is frequently used in catalytic processes as an alternative to improve enzyme activity and stability. Among the materials used as a support, nanomaterials have been successfully reported in the literature, mainly due to its high specific area. In this study, the nanomaterials graphene, reduced graphene oxide, boron nitride nanosheets, and gold nanoparticles were evaluated as a support for pepsin immobilization. The effectiveness of this process was evaluated by the content of immobilized protein and activity of immobilized pepsin, determined through casein hydrolysis, as well as circular dichroism to verify possible alterations in enzyme structure. The antioxidant properties of hydrolysates were assessed by DPPH, ABTS, FRAP, and β-carotene. All the materials studied presented a high efficiency of immobilization, with over 80% of the pepsin content bound to the support, the use of graphene derivatives as support led to a reduction in proteolytic activity, with the immobilized pepsin retaining only 6% of its activity. The 45 nm gold nanoparticle led to an increase in proteolytic activity from 0.050 to 0.104 U. No effect of amino group functionalization of boron nitride nanosheet was observed. The variations determined in the secondary structure, especially in the β-sheet content, corroborate the variations in activity after immobilization. The change in casein hydrolysates using pepsin immobilized in nanomaterials can be verified by reversed-phase chromatography. This change in the enzyme conformation affects the antioxidant properties of the casein hydrolysates. The results found in the present study contribute to the development of new biocatalysts based on the immobilization of enzymes in nanomaterials.</p>","PeriodicalId":17098,"journal":{"name":"Journal of separation science","volume":"49 10","pages":""},"PeriodicalIF":3.6,"publicationDate":"2026-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jssc.70546","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149146110","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Immobilization of Pepsin on Different Nanomaterials and Its Application in Obtaining Bioactive Peptides","authors":"Annie N. Alves,&nbsp;Márjorie C. P. Porfirio,&nbsp;Priscilla A. Nascimento,&nbsp;Jéssica F. Borges,&nbsp;Wenrong Yang,&nbsp;Motilal Mathesh,&nbsp;Paulo Bonomo,&nbsp;Rafael da C. I. Fontan,&nbsp;Renata C. F. Bonomo","doi":"10.1002/jssc.70546","DOIUrl":"https://doi.org/10.1002/jssc.70546","url":null,"abstract":"<p>Enzyme immobilization is frequently used in catalytic processes as an alternative to improve enzyme activity and stability. Among the materials used as a support, nanomaterials have been successfully reported in the literature, mainly due to its high specific area. In this study, the nanomaterials graphene, reduced graphene oxide, boron nitride nanosheets, and gold nanoparticles were evaluated as a support for pepsin immobilization. The effectiveness of this process was evaluated by the content of immobilized protein and activity of immobilized pepsin, determined through casein hydrolysis, as well as circular dichroism to verify possible alterations in enzyme structure. The antioxidant properties of hydrolysates were assessed by DPPH, ABTS, FRAP, and β-carotene. All the materials studied presented a high efficiency of immobilization, with over 80% of the pepsin content bound to the support, the use of graphene derivatives as support led to a reduction in proteolytic activity, with the immobilized pepsin retaining only 6% of its activity. The 45 nm gold nanoparticle led to an increase in proteolytic activity from 0.050 to 0.104 U. No effect of amino group functionalization of boron nitride nanosheet was observed. The variations determined in the secondary structure, especially in the β-sheet content, corroborate the variations in activity after immobilization. The change in casein hydrolysates using pepsin immobilized in nanomaterials can be verified by reversed-phase chromatography. This change in the enzyme conformation affects the antioxidant properties of the casein hydrolysates. The results found in the present study contribute to the development of new biocatalysts based on the immobilization of enzymes in nanomaterials.</p>","PeriodicalId":17098,"journal":{"name":"Journal of separation science","volume":"49 10","pages":""},"PeriodicalIF":3.6,"publicationDate":"2026-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jssc.70546","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149146123","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}