Oriented immobilization of enzymes helps to maintain their native structure and proper orientation for high-performance engineering to meet extensive biocatalysis demands.However,the supporting materials used for orie...Oriented immobilization of enzymes helps to maintain their native structure and proper orientation for high-performance engineering to meet extensive biocatalysis demands.However,the supporting materials used for orientated immobilization are usually costly or complicated in preparation,affecting their practical applications.In this work,a facile purification and immobilization method was proposed for enzyme immobilization based on organic-inorganic hybrid calcium phosphate nanocrystal(Ca Ps)induced by Cu^(2+) modified bovine serum albumin(BSA-Cu).Then,the as-prepared hybrid calcium phosphate nanosheet,BSA-Cu@Ca Ps,was utilized for one-pot purification and immobilization of His-tagged organophosphorus hydrolase(OPH)by metal-affinity binding to the incorporated BSA.BSA-Cu@Ca PsOPH exhibited enhanced p H stability and thermal stability compared to the free enzyme.Moreover,BSA-Cu@Ca Ps-OPH could retain more than 75%and 56%of initial activity after reuse 5 and 10 times,respectively.The results demonstrated that this facile strategy was promising for the effective biodegradation of organophosphorus pesticides with the immobilized enzyme.展开更多
Oriented ligand immobilization is one of the most effective strategies used in the design and construction of a high-capacity protein A chromatography. In this work, cysteine was introduced as anchoring sites by subst...Oriented ligand immobilization is one of the most effective strategies used in the design and construction of a high-capacity protein A chromatography. In this work, cysteine was introduced as anchoring sites by substituting a specific residue on Helix Ⅰ, Ⅱ, and at C-terminus of antibody binding domain Z from protein A, respectively, to investigate structural evolution and binding behavior of protein A ligands at liquid-solid interfaces. Among the three affinity dextran-coated Fe_(3)O_(4) magnetic nanoparticles(Fe_(3)O_(4)@Dx MNPs), affinity MNPs with the immobilized ligand via N11C on Helix Ⅰ(Fe_(3)O_(4)@Dx-Z_(1) MNPs) had the highest helical content, and MNPs with the immobilized ligand via G29C on Helix Ⅱ(Fe_(3)O_(4)@Dx-Z_(2) MNPs) had the lowest helical content at the same pHs. It was attributed to less electrostatic attraction of ligand to negatively charged surface on Fe_(3)O_(4)@Dx-Z_(1) MNPs because of less positive charged residues on Helix Ⅰ(K6) than Helix Ⅱ(R27/K35). Among the three affinity MNPs, moreover, the highest affinity to immunoglobulin G(IgG) binding was observed on Fe_(3)O_(4)@Dx-Z_(1) MNPs in isothermal titration calorimetry measurement, further validating greater structural integrity of the ligand on Fe_(3)O_(4)@Dx-Z_(1) MNPs. Finally,the study of IgG binding on MNPs and 96-well plates showed that anchoring sites for ligand immobilization had distinct influences on IgG binding and IgG-mediated antigen binding. This work illustrated that anchoring sites of the ligands had a striking significance for the molecular structure of the ligand at liquid-solid interfaces and raised an important implication for the design and optimization of protein A chromatography and protein A-based immunoassay analysis.展开更多
We described a novel polymer-lipase conjugate for high-efficient esterification of vitamin E using vitamin E and succinic anhydride as the substrates in nonaqueous media.In this work,the monomer,N-isopropylacrylamide(...We described a novel polymer-lipase conjugate for high-efficient esterification of vitamin E using vitamin E and succinic anhydride as the substrates in nonaqueous media.In this work,the monomer,N-isopropylacrylamide(NIPAM),was grafted onto Candida rugosa lipase(CRL)to synthesize poly(NIPAM)(pNIPAM)-CRL conjugate by atom transfer radical polymerization via the initiator coupled on the surface of CRL.The result showed that the catalytic efficiencies of pNIPAM-CRL conjugates(19.5-30.3 L·s^(-1)·mmol^(-1))were at least 7 times higher than that of free CRL(2.36 L·s^(-1)·mmol^(-1))in DMSO.It was attributed to a significant increase in Kcat of the conjugates in nonaqueous media.The synthesis catalyzed by pNIPAM-CRL co njugates was influenced by the length and density of the grafted polymer,water content,solvent polarity and molar ratio of the substrates.In the optimal synthesis,the reaction time was shortened at least 7 times,and yields of vitamin E succinate by pNIPAM-g-CRL and free CRL were obtained to be 75.4%and 6.6%at 55℃after the reaction for 1.5 h.The result argued that conjugation with pNIPAM induced conformational change of the lid on CRL based on hydrophobic interaction,thus providing a higher possibility of catalysis-favorable conformation on CRL in nonaqueous media.Moreover,pNIPAM conjugation improved the thermal stability of CRL greatly,and the stability improved further with an increase of chain length of pNIPAM.At the optimal reaction conditions(55℃and 1.5 h),pNIPAM-g-CRL also exhibited good reusability in the enzymatic synthesis of vitamin E succinate and kept~70%of its catalytic activity after ten consecutive cycles.The research demonstrated that pNIPAM-g-CRL was a more competitive biocatalyst in the enzymatic synthesis of vitamin E succinate and exhibited good application potential under harsh industrial conditions.展开更多
Polymer-grafted ion exchange adsorbents were of great interest for the development of high-performance protein chromatography in biopharmaceutical and related fields.In this work,protein retention was systematically i...Polymer-grafted ion exchange adsorbents were of great interest for the development of high-performance protein chromatography in biopharmaceutical and related fields.In this work,protein retention was systematically investigated in ion exchange chromatography packed respectively with dextran-grafted cation exchange adsorbents containing sulphopropyl(SP)ligand,SP Sepharose XL and Capto S,and non-grafted cation exchange adsorbent,SP Sepharose FF,using five proteins.With an increase of buffer p Hs,retention factors of proteins decreased among all the adsorbents,demonstrating the dominant role of electrostatic interaction for protein binding on cation exchange adsorbents.The evidences further revealed that the scattered positive charges on the surface of protein molecules,rather than net charge of protein molecule,determined protein retention on cation exchange adsorbent.Likely,counterions including NH4^+,K^+,Na^+and Mg^2+exhibited distinct influence on protein retention.It was well ascribed to solvent-mediated indirect ion-macromolecule interactions and direct ion-macromolecule interactions.Compared with SP Sepharose FF,polymer structure in dextran-grafted cation exchange adsorbents ultimately brought about different ligand distributions and smaller pore sizes,thereby regulating protein retention in cation exchange chromatography.By comparing the retention of myoglobin andβ-lactoglobulin B in SP Sepharose XL and Capto S,we reasonably speculated that the enhancement of nonelectrostatic interaction caused by reducing the space arm length was a major reason for an increasing retention factor of myoglobin in Capto S.The results in this research help us understand adsorption mechanism of protein in polymer-grafted adsorbents and give scientific guidance for the development of chromatographic materials.展开更多
Protein A chromatography is a key technology in the industrial production of antibodies,and a variety of commercial protein A adsorbents are available in shelf.High stability and binding capacity of a protein A adsorb...Protein A chromatography is a key technology in the industrial production of antibodies,and a variety of commercial protein A adsorbents are available in shelf.High stability and binding capacity of a protein A adsorbent are two key issues for successful practice of protein A chromatography.Earlier versions of protein A adsorbents ever exhibited serious fragility to typical cleaning-in-place protocols(e.g.washing with sodium hydroxide solution),and suffered from low binding capacity,harsh elution,ligand leakage and other problems involved in industrial applications.During the last three decades,various techniques and approaches have been applied in the improvement of chemical stability and enhancement of binding capacity of protein A-based ligands and adsorbents for antibody purifications.This mini-review focuses on the technical explorations in protein A-based affinity adsorbents,especially protein A-based ligands,including the efforts to increase the chemical stability by site-directed mutations and to improve the binding capacity by ligand polymerization and site-directed immobilization.Moreover,the efforts to develop short peptide ligands based on the structure of protein A,including the biomimetic design strategies and the synthesis of peptide-mixed mode hybrid ligands are discussed.These peptide and peptidebased hybrid ligands exhibit high affinity and selectivity to antibodies,but noteworthy differences in the binding mechanism of antibody from protein A.As a result,bound antibody to the ligands could be effectively eluted under mild conditions.Perspectives for the development of the protein A-based peptide ligands have been extensively discussed,suggesting that the ligands represent a direction for technological development of antibody purification.展开更多
In this work, we have synthesized two polymer-grafted cation exchangers: one via the grafting-from approach, in which sulfopropyl methacrylate (SPM) is grafted through atom transfer radical polymerization onto Sepharo...In this work, we have synthesized two polymer-grafted cation exchangers: one via the grafting-from approach, in which sulfopropyl methacrylate (SPM) is grafted through atom transfer radical polymerization onto Sepharose FF (the thus resulting exchanger is referred as Sep-g-SPM), and another via the grafting-to approach, in which the polymer of SPM is directly coupled onto Sepharose FF (the thus resulting exchanger is called as Sep-pSPM). Protein adsorption on these two cation exchangers have been also investigated. At the same ligand density, Sep-g-SPM has a larger accessible pore radius and a smaller depth of polymer layer than Sep-pSPM, due to the controllable introduction of polymer chains with the regular distribution of the ligand. Therefore, high-capacity adsorption of lysozyme and γ-globulin could be achieved simultaneously in Sep-g-SPM with an ionic capacity (IC) of 308 mmol·L^-1. However, Sep-pSPM has an irregular chain distribution and different architecture of polymer layer, which lead to more serious repulsive interaction to proteins, and thus Sep-pSPM has a lower adsorption capacity for γ-globulin than Sep-g-SPM with the similar IC. Moreover, the results from protein uptake experiments indicate that the facilitated transport of adsorbed γ-globulin occurs only in Sep-pSPM and depends on the architecture of polymer layers. Our research provides a clear clue for the development of high-performance protein chromatography.展开更多
Inspired by nature,precise spatial organization of enzyme cascades of interest is crucial to the improvement of catalytic performance.Herein,DNA scaffolds were introduced to construct a toolkit for versatile immobiliz...Inspired by nature,precise spatial organization of enzyme cascades of interest is crucial to the improvement of catalytic performance.Herein,DNA scaffolds were introduced to construct a toolkit for versatile immobilization of enzyme pairs on dextran-coated magnetic nanoparticles(MNPs).After the glucose oxidase(GOx)and horseradish peroxidase(HRP)pair was immobilized through random cova-lent,DNA-directed and DNA tile-directed strategies,the immobilized GOx/HRP pair on the MNP-based carrier assembled with DNA tile(TD@MNPs)exhibited the highest activity due to rational spatial organization and less conformational change of constituent enzymes.With a decrease in interenzyme distance on TD@MNPs,furthermore,the catalytic efficiency of the HRP/GOx pair increased further for both substrates,2,2'-azinobis(3-ethyl-benzthiazoline-6-sulfonate)(ABTS)and 3,3',5,5'-tetramethyl benzidine(TMB).As the assembled HRP was closer to the carrier surface,the catalytic efficiency of the GOx/HRP pair increased by 6.2-fold for positively charged TMB and only by 62%for negatively charged ABTS compared with the free GOx/HRP pair.Moreover,a reversal of catalytic efficiency was found after the GOx/HRP pair was assembled on a positively charged carrier(TD@pMNPs).This research demonstrated that MNP-based car-riers had the potential to become a versatile toolkit for shedding an insight into catalytic performance and the development of new biocatalysts.展开更多
基金supported by the National Key Research and Development Program of China(2021YFC2102801)the National Natural Science Foundation of China(21621004)。
文摘Oriented immobilization of enzymes helps to maintain their native structure and proper orientation for high-performance engineering to meet extensive biocatalysis demands.However,the supporting materials used for orientated immobilization are usually costly or complicated in preparation,affecting their practical applications.In this work,a facile purification and immobilization method was proposed for enzyme immobilization based on organic-inorganic hybrid calcium phosphate nanocrystal(Ca Ps)induced by Cu^(2+) modified bovine serum albumin(BSA-Cu).Then,the as-prepared hybrid calcium phosphate nanosheet,BSA-Cu@Ca Ps,was utilized for one-pot purification and immobilization of His-tagged organophosphorus hydrolase(OPH)by metal-affinity binding to the incorporated BSA.BSA-Cu@Ca PsOPH exhibited enhanced p H stability and thermal stability compared to the free enzyme.Moreover,BSA-Cu@Ca Ps-OPH could retain more than 75%and 56%of initial activity after reuse 5 and 10 times,respectively.The results demonstrated that this facile strategy was promising for the effective biodegradation of organophosphorus pesticides with the immobilized enzyme.
基金supported by the National Natural Science Foundation of China (Nos. 21878221 and 21476166)the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (No. 21621004)。
文摘Oriented ligand immobilization is one of the most effective strategies used in the design and construction of a high-capacity protein A chromatography. In this work, cysteine was introduced as anchoring sites by substituting a specific residue on Helix Ⅰ, Ⅱ, and at C-terminus of antibody binding domain Z from protein A, respectively, to investigate structural evolution and binding behavior of protein A ligands at liquid-solid interfaces. Among the three affinity dextran-coated Fe_(3)O_(4) magnetic nanoparticles(Fe_(3)O_(4)@Dx MNPs), affinity MNPs with the immobilized ligand via N11C on Helix Ⅰ(Fe_(3)O_(4)@Dx-Z_(1) MNPs) had the highest helical content, and MNPs with the immobilized ligand via G29C on Helix Ⅱ(Fe_(3)O_(4)@Dx-Z_(2) MNPs) had the lowest helical content at the same pHs. It was attributed to less electrostatic attraction of ligand to negatively charged surface on Fe_(3)O_(4)@Dx-Z_(1) MNPs because of less positive charged residues on Helix Ⅰ(K6) than Helix Ⅱ(R27/K35). Among the three affinity MNPs, moreover, the highest affinity to immunoglobulin G(IgG) binding was observed on Fe_(3)O_(4)@Dx-Z_(1) MNPs in isothermal titration calorimetry measurement, further validating greater structural integrity of the ligand on Fe_(3)O_(4)@Dx-Z_(1) MNPs. Finally,the study of IgG binding on MNPs and 96-well plates showed that anchoring sites for ligand immobilization had distinct influences on IgG binding and IgG-mediated antigen binding. This work illustrated that anchoring sites of the ligands had a striking significance for the molecular structure of the ligand at liquid-solid interfaces and raised an important implication for the design and optimization of protein A chromatography and protein A-based immunoassay analysis.
基金financially supported by the National Key Research and Development Program of China (2021YFC2102801)National Natural Science Foundation of China (21878221)+1 种基金the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (21621004)the Haihe Laboratory of Sustainable Chemical Transformations for financial support.
文摘We described a novel polymer-lipase conjugate for high-efficient esterification of vitamin E using vitamin E and succinic anhydride as the substrates in nonaqueous media.In this work,the monomer,N-isopropylacrylamide(NIPAM),was grafted onto Candida rugosa lipase(CRL)to synthesize poly(NIPAM)(pNIPAM)-CRL conjugate by atom transfer radical polymerization via the initiator coupled on the surface of CRL.The result showed that the catalytic efficiencies of pNIPAM-CRL conjugates(19.5-30.3 L·s^(-1)·mmol^(-1))were at least 7 times higher than that of free CRL(2.36 L·s^(-1)·mmol^(-1))in DMSO.It was attributed to a significant increase in Kcat of the conjugates in nonaqueous media.The synthesis catalyzed by pNIPAM-CRL co njugates was influenced by the length and density of the grafted polymer,water content,solvent polarity and molar ratio of the substrates.In the optimal synthesis,the reaction time was shortened at least 7 times,and yields of vitamin E succinate by pNIPAM-g-CRL and free CRL were obtained to be 75.4%and 6.6%at 55℃after the reaction for 1.5 h.The result argued that conjugation with pNIPAM induced conformational change of the lid on CRL based on hydrophobic interaction,thus providing a higher possibility of catalysis-favorable conformation on CRL in nonaqueous media.Moreover,pNIPAM conjugation improved the thermal stability of CRL greatly,and the stability improved further with an increase of chain length of pNIPAM.At the optimal reaction conditions(55℃and 1.5 h),pNIPAM-g-CRL also exhibited good reusability in the enzymatic synthesis of vitamin E succinate and kept~70%of its catalytic activity after ten consecutive cycles.The research demonstrated that pNIPAM-g-CRL was a more competitive biocatalyst in the enzymatic synthesis of vitamin E succinate and exhibited good application potential under harsh industrial conditions.
基金supported by the National Natural Science Foundation of China(Nos.21476166 and 21878221)the Foundation for Innovative Research Groups of the National Natural Science Foundation of China(No.21621004)。
文摘Polymer-grafted ion exchange adsorbents were of great interest for the development of high-performance protein chromatography in biopharmaceutical and related fields.In this work,protein retention was systematically investigated in ion exchange chromatography packed respectively with dextran-grafted cation exchange adsorbents containing sulphopropyl(SP)ligand,SP Sepharose XL and Capto S,and non-grafted cation exchange adsorbent,SP Sepharose FF,using five proteins.With an increase of buffer p Hs,retention factors of proteins decreased among all the adsorbents,demonstrating the dominant role of electrostatic interaction for protein binding on cation exchange adsorbents.The evidences further revealed that the scattered positive charges on the surface of protein molecules,rather than net charge of protein molecule,determined protein retention on cation exchange adsorbent.Likely,counterions including NH4^+,K^+,Na^+and Mg^2+exhibited distinct influence on protein retention.It was well ascribed to solvent-mediated indirect ion-macromolecule interactions and direct ion-macromolecule interactions.Compared with SP Sepharose FF,polymer structure in dextran-grafted cation exchange adsorbents ultimately brought about different ligand distributions and smaller pore sizes,thereby regulating protein retention in cation exchange chromatography.By comparing the retention of myoglobin andβ-lactoglobulin B in SP Sepharose XL and Capto S,we reasonably speculated that the enhancement of nonelectrostatic interaction caused by reducing the space arm length was a major reason for an increasing retention factor of myoglobin in Capto S.The results in this research help us understand adsorption mechanism of protein in polymer-grafted adsorbents and give scientific guidance for the development of chromatographic materials.
基金This work was supported by the National Natural Science Foundation of China(Nos.21476166 and 21878221)the Foundation for Innovative Research Groups of the National Natural Science Foundation of China(No.21621004).
文摘Protein A chromatography is a key technology in the industrial production of antibodies,and a variety of commercial protein A adsorbents are available in shelf.High stability and binding capacity of a protein A adsorbent are two key issues for successful practice of protein A chromatography.Earlier versions of protein A adsorbents ever exhibited serious fragility to typical cleaning-in-place protocols(e.g.washing with sodium hydroxide solution),and suffered from low binding capacity,harsh elution,ligand leakage and other problems involved in industrial applications.During the last three decades,various techniques and approaches have been applied in the improvement of chemical stability and enhancement of binding capacity of protein A-based ligands and adsorbents for antibody purifications.This mini-review focuses on the technical explorations in protein A-based affinity adsorbents,especially protein A-based ligands,including the efforts to increase the chemical stability by site-directed mutations and to improve the binding capacity by ligand polymerization and site-directed immobilization.Moreover,the efforts to develop short peptide ligands based on the structure of protein A,including the biomimetic design strategies and the synthesis of peptide-mixed mode hybrid ligands are discussed.These peptide and peptidebased hybrid ligands exhibit high affinity and selectivity to antibodies,but noteworthy differences in the binding mechanism of antibody from protein A.As a result,bound antibody to the ligands could be effectively eluted under mild conditions.Perspectives for the development of the protein A-based peptide ligands have been extensively discussed,suggesting that the ligands represent a direction for technological development of antibody purification.
基金the National Natural Science Foundation of China (Grant Nos. 21476166 and 21236005)the Open Funding Project of the State Key Laboratory of Biochemical Engineering (No. 2014KF-03)the Tianjin Natural Science Foundation (15JCYBJC48500).
文摘In this work, we have synthesized two polymer-grafted cation exchangers: one via the grafting-from approach, in which sulfopropyl methacrylate (SPM) is grafted through atom transfer radical polymerization onto Sepharose FF (the thus resulting exchanger is referred as Sep-g-SPM), and another via the grafting-to approach, in which the polymer of SPM is directly coupled onto Sepharose FF (the thus resulting exchanger is called as Sep-pSPM). Protein adsorption on these two cation exchangers have been also investigated. At the same ligand density, Sep-g-SPM has a larger accessible pore radius and a smaller depth of polymer layer than Sep-pSPM, due to the controllable introduction of polymer chains with the regular distribution of the ligand. Therefore, high-capacity adsorption of lysozyme and γ-globulin could be achieved simultaneously in Sep-g-SPM with an ionic capacity (IC) of 308 mmol·L^-1. However, Sep-pSPM has an irregular chain distribution and different architecture of polymer layer, which lead to more serious repulsive interaction to proteins, and thus Sep-pSPM has a lower adsorption capacity for γ-globulin than Sep-g-SPM with the similar IC. Moreover, the results from protein uptake experiments indicate that the facilitated transport of adsorbed γ-globulin occurs only in Sep-pSPM and depends on the architecture of polymer layers. Our research provides a clear clue for the development of high-performance protein chromatography.
基金the National Key Research and Development Program of China(No.2021YFC2102801)the National Natural Science Foundation of China(No.21878221)the Foundation for Innovative Research Groups of the National Natural Science Foundation of China(No.21621004).
文摘Inspired by nature,precise spatial organization of enzyme cascades of interest is crucial to the improvement of catalytic performance.Herein,DNA scaffolds were introduced to construct a toolkit for versatile immobilization of enzyme pairs on dextran-coated magnetic nanoparticles(MNPs).After the glucose oxidase(GOx)and horseradish peroxidase(HRP)pair was immobilized through random cova-lent,DNA-directed and DNA tile-directed strategies,the immobilized GOx/HRP pair on the MNP-based carrier assembled with DNA tile(TD@MNPs)exhibited the highest activity due to rational spatial organization and less conformational change of constituent enzymes.With a decrease in interenzyme distance on TD@MNPs,furthermore,the catalytic efficiency of the HRP/GOx pair increased further for both substrates,2,2'-azinobis(3-ethyl-benzthiazoline-6-sulfonate)(ABTS)and 3,3',5,5'-tetramethyl benzidine(TMB).As the assembled HRP was closer to the carrier surface,the catalytic efficiency of the GOx/HRP pair increased by 6.2-fold for positively charged TMB and only by 62%for negatively charged ABTS compared with the free GOx/HRP pair.Moreover,a reversal of catalytic efficiency was found after the GOx/HRP pair was assembled on a positively charged carrier(TD@pMNPs).This research demonstrated that MNP-based car-riers had the potential to become a versatile toolkit for shedding an insight into catalytic performance and the development of new biocatalysts.
