{"title":"揭示柠檬酸单、二和三胆铵-乙二醇 DES 对α-糜蛋白酶的稳定机制,以保存和激活该酶","authors":"Bindu Yadav, Niketa Yadav, Pannuru Venkatesu","doi":"10.1039/d4cp03315a","DOIUrl":null,"url":null,"abstract":"Deep eutectic solvents (DESs) are considered as designer solvents which serve as an alternative to traditional solvents. Numerous favourable properties and advantageous characteristics promote their utility in bio-catalysis. Therefore, these have emerged as attractive sustainable media for different biomacromolecules. In the present work, we have synthesized cholinium- based DESs having molar ratio of hydrogen bond acceptor (HBA): hydrogen bond donor (HBD) in 1:2 by varying the cationic ratio in HBA forming the DESs as monocholinium citrate ([Chn][Cit]), dicholinium citrate ([Chn]2[Cit]) and tricholinium citrate ([Chn]3[Cit]), keeping HBD constant as ethylene glycol (EG) to study their suitability for α-chymotrypsin (α-CT). Herein, we have systematically evaluated the influence of DES-1 ([Chn][Cit])-[EG]), DES-2 ([Chn]2[Cit])-[EG]) and DES-3 ([Chn]3[Cit])-[EG]) on structural and thermal stability, thermodynamic profile, colloidal stability and enzymatic activity of α-CT using different spectroscopic techniques. The spectroscopic results explicitly elucidate enhanced structural stability and activity of enzyme as the cationic ratio on HBA increases. Fascinatingly, temperature-dependent studies through both fluorescence and activity measurements supported that DES-2 and DES-3 have highly beneficial effects on α-CT stability. The transition temperature (Tm) of α-CT was augmented by 12 °C in DES-2, 10 °C in DES-3 and 9.1 °C in DES-1 when compared to the enzyme in buffer. Furthermore, transmission electron microscopy (TEM) analysis revealed that the morphology of α-CT in DES-2 and DES-3 closely mirrored the structure of α-CT, while DES-1 exhibited only minor structural deviations. These findings were corroborated by hydrodynamic size (dH) measurements and average decay time analysis, which confirmed the observed morphological similarities and perturbations. The long-term preservation ability and kinetics of DES-3 was eventually confirmed by Michaelis-Menten kinetics. Ultimately, these outcomes demonstrate that by increasing the molar ratio of cholinium cation in HBA can lead to intensify the proficiency of DESs to stabilize the α-CT structure. Our results also suggest that the effect imparted by DESs was due to DES itself rather than its composing elements. Also, how the biocompatibility of DESs towards enzymes can be varied by changing molar ratios of the constituent components of DESs to facilitate the expansion of applicability of DESs in biocatalysis.","PeriodicalId":99,"journal":{"name":"Physical Chemistry Chemical Physics","volume":null,"pages":null},"PeriodicalIF":2.9000,"publicationDate":"2024-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Unravelling the stabilization mechanism of mono-, di and tri-cholinium citrate-ethylene glycol DESs towards α-chymotrypsin for preservation and activation of the enzyme\",\"authors\":\"Bindu Yadav, Niketa Yadav, Pannuru Venkatesu\",\"doi\":\"10.1039/d4cp03315a\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"Deep eutectic solvents (DESs) are considered as designer solvents which serve as an alternative to traditional solvents. Numerous favourable properties and advantageous characteristics promote their utility in bio-catalysis. Therefore, these have emerged as attractive sustainable media for different biomacromolecules. In the present work, we have synthesized cholinium- based DESs having molar ratio of hydrogen bond acceptor (HBA): hydrogen bond donor (HBD) in 1:2 by varying the cationic ratio in HBA forming the DESs as monocholinium citrate ([Chn][Cit]), dicholinium citrate ([Chn]2[Cit]) and tricholinium citrate ([Chn]3[Cit]), keeping HBD constant as ethylene glycol (EG) to study their suitability for α-chymotrypsin (α-CT). Herein, we have systematically evaluated the influence of DES-1 ([Chn][Cit])-[EG]), DES-2 ([Chn]2[Cit])-[EG]) and DES-3 ([Chn]3[Cit])-[EG]) on structural and thermal stability, thermodynamic profile, colloidal stability and enzymatic activity of α-CT using different spectroscopic techniques. The spectroscopic results explicitly elucidate enhanced structural stability and activity of enzyme as the cationic ratio on HBA increases. Fascinatingly, temperature-dependent studies through both fluorescence and activity measurements supported that DES-2 and DES-3 have highly beneficial effects on α-CT stability. The transition temperature (Tm) of α-CT was augmented by 12 °C in DES-2, 10 °C in DES-3 and 9.1 °C in DES-1 when compared to the enzyme in buffer. Furthermore, transmission electron microscopy (TEM) analysis revealed that the morphology of α-CT in DES-2 and DES-3 closely mirrored the structure of α-CT, while DES-1 exhibited only minor structural deviations. These findings were corroborated by hydrodynamic size (dH) measurements and average decay time analysis, which confirmed the observed morphological similarities and perturbations. The long-term preservation ability and kinetics of DES-3 was eventually confirmed by Michaelis-Menten kinetics. Ultimately, these outcomes demonstrate that by increasing the molar ratio of cholinium cation in HBA can lead to intensify the proficiency of DESs to stabilize the α-CT structure. Our results also suggest that the effect imparted by DESs was due to DES itself rather than its composing elements. Also, how the biocompatibility of DESs towards enzymes can be varied by changing molar ratios of the constituent components of DESs to facilitate the expansion of applicability of DESs in biocatalysis.\",\"PeriodicalId\":99,\"journal\":{\"name\":\"Physical Chemistry Chemical Physics\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":2.9000,\"publicationDate\":\"2024-10-22\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Physical Chemistry Chemical Physics\",\"FirstCategoryId\":\"92\",\"ListUrlMain\":\"https://doi.org/10.1039/d4cp03315a\",\"RegionNum\":3,\"RegionCategory\":\"化学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"CHEMISTRY, PHYSICAL\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Physical Chemistry Chemical Physics","FirstCategoryId":"92","ListUrlMain":"https://doi.org/10.1039/d4cp03315a","RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"CHEMISTRY, PHYSICAL","Score":null,"Total":0}
Unravelling the stabilization mechanism of mono-, di and tri-cholinium citrate-ethylene glycol DESs towards α-chymotrypsin for preservation and activation of the enzyme
Deep eutectic solvents (DESs) are considered as designer solvents which serve as an alternative to traditional solvents. Numerous favourable properties and advantageous characteristics promote their utility in bio-catalysis. Therefore, these have emerged as attractive sustainable media for different biomacromolecules. In the present work, we have synthesized cholinium- based DESs having molar ratio of hydrogen bond acceptor (HBA): hydrogen bond donor (HBD) in 1:2 by varying the cationic ratio in HBA forming the DESs as monocholinium citrate ([Chn][Cit]), dicholinium citrate ([Chn]2[Cit]) and tricholinium citrate ([Chn]3[Cit]), keeping HBD constant as ethylene glycol (EG) to study their suitability for α-chymotrypsin (α-CT). Herein, we have systematically evaluated the influence of DES-1 ([Chn][Cit])-[EG]), DES-2 ([Chn]2[Cit])-[EG]) and DES-3 ([Chn]3[Cit])-[EG]) on structural and thermal stability, thermodynamic profile, colloidal stability and enzymatic activity of α-CT using different spectroscopic techniques. The spectroscopic results explicitly elucidate enhanced structural stability and activity of enzyme as the cationic ratio on HBA increases. Fascinatingly, temperature-dependent studies through both fluorescence and activity measurements supported that DES-2 and DES-3 have highly beneficial effects on α-CT stability. The transition temperature (Tm) of α-CT was augmented by 12 °C in DES-2, 10 °C in DES-3 and 9.1 °C in DES-1 when compared to the enzyme in buffer. Furthermore, transmission electron microscopy (TEM) analysis revealed that the morphology of α-CT in DES-2 and DES-3 closely mirrored the structure of α-CT, while DES-1 exhibited only minor structural deviations. These findings were corroborated by hydrodynamic size (dH) measurements and average decay time analysis, which confirmed the observed morphological similarities and perturbations. The long-term preservation ability and kinetics of DES-3 was eventually confirmed by Michaelis-Menten kinetics. Ultimately, these outcomes demonstrate that by increasing the molar ratio of cholinium cation in HBA can lead to intensify the proficiency of DESs to stabilize the α-CT structure. Our results also suggest that the effect imparted by DESs was due to DES itself rather than its composing elements. Also, how the biocompatibility of DESs towards enzymes can be varied by changing molar ratios of the constituent components of DESs to facilitate the expansion of applicability of DESs in biocatalysis.
期刊介绍:
Physical Chemistry Chemical Physics (PCCP) is an international journal co-owned by 19 physical chemistry and physics societies from around the world. This journal publishes original, cutting-edge research in physical chemistry, chemical physics and biophysical chemistry. To be suitable for publication in PCCP, articles must include significant innovation and/or insight into physical chemistry; this is the most important criterion that reviewers and Editors will judge against when evaluating submissions.
The journal has a broad scope and welcomes contributions spanning experiment, theory, computation and data science. Topical coverage includes spectroscopy, dynamics, kinetics, statistical mechanics, thermodynamics, electrochemistry, catalysis, surface science, quantum mechanics, quantum computing and machine learning. Interdisciplinary research areas such as polymers and soft matter, materials, nanoscience, energy, surfaces/interfaces, and biophysical chemistry are welcomed if they demonstrate significant innovation and/or insight into physical chemistry. Joined experimental/theoretical studies are particularly appreciated when complementary and based on up-to-date approaches.