Herein,we report the facile conjugation between proteins and water-soluble [60]fullerene derivatives(DC_(60)) under native conditions using SpyTag as a reactive handle.Water-soluble [60] fullerene derivatives were fir...Herein,we report the facile conjugation between proteins and water-soluble [60]fullerene derivatives(DC_(60)) under native conditions using SpyTag as a reactive handle.Water-soluble [60] fullerene derivatives were first prepared via sequential Bingel-Hirsch reaction and "clicked" with SpyTag to give DC_(60)-SpyTag for native conjugation with proteins by the highly efficient SpyTag-SpyCatcher chemistry.The bioconjugation was confirmed by MALDI-TOF MS spectra and SDS-PAGE analysis.The TEM and UVvis spectroscopic study further revealed that the DC_(60) could alter the optical performance and induce aggregation of the target proteins.It thus provides a general and robust method for modifying proteins with C_(60) derivatives and could potentially be adapted for native conjugation between proteins and other nonbiological motifs as well.展开更多
Laccases,belonging to the superfamily of multicopper oxidases,can perform electron oxidation on a broad range of substrates,releasing only water as a by-product.Although instability and aggregation significantly const...Laccases,belonging to the superfamily of multicopper oxidases,can perform electron oxidation on a broad range of substrates,releasing only water as a by-product.Although instability and aggregation significantly constrain the industrial use of these eco-friendly biocatalysts,it is a daunting challenge for current engineering strategies to elevate these crucial enzymatic characteristics simultaneously.Here,we developed a cyclizing laccase(CyLacc)using SpyTag/SpyCatcher technology,which endows the enzyme with high thermostability and high solubility.Differential scanning calorimetry sheds light on the thermal unfolding and refolding processes of CyLacc,indicating its increased tolerance to high-temperature environments.Furthermore,utilizing large-scale molecular dynamics simulations,we found that the reduction of the hydrophobic surface and the increased flexibility of the loops are the main reasons for the increase in solubility.Compared with the wild-type laccase,the improved features led to a much higher alkaline lignin,Malachite Green,and Neutral Red decolorization efficiency of CyLacc.Furthermore,CyLacc significantly enhanced the polymerization yields of catechol and hydroquinone,from 49%and 63.6%(with wild-type Lacc)to 78.5%and 90.3%,respectively.The number-average molecular weights of the polyphenols ranged from 1,000 to 1,200 D(corresponding to the degree of polymerization that varied from 10 to 13),showing selective polymerization of phenolic compounds catalyzed by laccase.The present work,combining theory with experiment,opens up a new avenue for industrially important laccase engineering and is expected to contribute to lignin valorization and environmental protection.展开更多
Synthesis of macromolecular systems with precise structural and functional control constitutes a fundamental challenge for materials science and engineering. Development of the ability to construct complex bio-macromo...Synthesis of macromolecular systems with precise structural and functional control constitutes a fundamental challenge for materials science and engineering. Development of the ability to construct complex bio-macromolecular architectures provides a solution to this challenge. The past few years have witnessed the emergence of a new category of peptide-protein chemistry which can covalently stitch together protein]peptide molecules with high specificity under mild physiological conditions. It has thus inspired the concept of genetically encoded click chemistry （GECC）. As a prototype of GECC, SpyTag/ SpyCatcher chemistry has enabled the precise synthesis ofmacromolecules both in vitro and in vivo, exerting precise control over the fundamental properties of these macromolecules including length, sequence, stereochemistry and topology and leading to the creation of diverse biomaterials for a variety of applications. We thus anticipate a potential toolbox of GECC comprising multiple mutually orthogonal, covalent-bond forming peptide-protein reactive pairs with diverse features, which shall bridge synthetic biology and materials science and open up enormous opportunities for biomaterialsin the future.展开更多
Due to the complexity of bioactive ingredients in biological samples,the screening of target proteins is a complex process.Herein,a feasible strategy for directing protein immobilization on silica magnetic beads for l...Due to the complexity of bioactive ingredients in biological samples,the screening of target proteins is a complex process.Herein,a feasible strategy for directing protein immobilization on silica magnetic beads for ligand fishing based on SpyTag/SpyCatcher(ST/SC)-mediated anchoring is presented.Carboxyl functional groups on the surface of silica-coated magnetic beads(SMBs)were coupled with SC using the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride/N-hydroxysulfosuccinimide method,named SC-SMBs.The green fluorescent protein(GFP),as the capturing protein model,was ST-labeled and anchored at a specific orientation onto the surface of SC-SMBs directly from relevant cell lysates via ST/SC self-ligation.The characteristics of the SC-SMBs were studied via electron microscopy,energy dispersive spectroscopy,and Fourier transform infrared spectroscopy.The spontaneity and site-specificity of this unique reaction were confirmed via electrophoresis and fluorescence analyses.Although the alkaline stability of ST-GFP-ligated SC-SMBs was not ideal,the formed isopeptide bond was unbreakable under acidic conditions(0.05 M glycine-HCl buffer,pH 1e6)for 2 h,under 20%ethanol solution within 7 days,and at most temperatures.We,therefore,present a simple and universal strategy for the preparation of diverse protein-functionalized SMBs for ligand fishing,prompting its usage on drug screening and target finding.展开更多
Allitol is a valuable drug intermediate and a potential low-calorie sweetener in food industry.However,developing an efficient biotransformation system for large-scale production of allitol remains challenging.Here,a ...Allitol is a valuable drug intermediate and a potential low-calorie sweetener in food industry.However,developing an efficient biotransformation system for large-scale production of allitol remains challenging.Here,a customized multienzyme cascade immobilization system was designed for efficient biocatalytic synthesis of allitol from D-fructose.The system included three immobilized enzymes:amino resin(LX1000)-immobilized Dallulose 3-epimerase(SfDAE)from Sinorhizobium fredii,LX1000-immobilized ribitol dehydrogenase(KpRDH)from Klebsiella pneumoniae,and derived formate dehydrogenase(CbFDH)from Candida boidinii was directional immobilization on bacterial cellulose(BC)employ SpyCatcher/SpyTag modular.By employing a strategy of directional immobilization and SpyCatcher-functionalized BC,the poor cascade cooperativity in the rate-limiting enzyme(CbFDH)was successfully resolved.Under the optimum biotransformation conditions,this system was able to convert 500 g/L of D-fructose into 455 g/L of allitol with conversion yields as high as 90%.Importantly,they also had excellent stability,retaining 60%relative activity after 10 repeated cycles,and preserving 50.2%relative activity after 24 days of storage.Consequently,the design and application of this multienzyme cascade immobilization system successfully provides an effective strategy for the large-scale biosynthesis of allitol.展开更多
基金the financial support from the National Natural Science Foundation of China(Nos.21925102,21991132 and 21674003)supported by Beijing National Laboratory for Molecular Sciences(No.BNLMS-CXXM-202006)Clinical Medicine Plus X Project of Peking University,Fundamental Research Funds for the Central Universities。
文摘Herein,we report the facile conjugation between proteins and water-soluble [60]fullerene derivatives(DC_(60)) under native conditions using SpyTag as a reactive handle.Water-soluble [60] fullerene derivatives were first prepared via sequential Bingel-Hirsch reaction and "clicked" with SpyTag to give DC_(60)-SpyTag for native conjugation with proteins by the highly efficient SpyTag-SpyCatcher chemistry.The bioconjugation was confirmed by MALDI-TOF MS spectra and SDS-PAGE analysis.The TEM and UVvis spectroscopic study further revealed that the DC_(60) could alter the optical performance and induce aggregation of the target proteins.It thus provides a general and robust method for modifying proteins with C_(60) derivatives and could potentially be adapted for native conjugation between proteins and other nonbiological motifs as well.
基金supported by the National Key Research and Development Program of China(2023YFC3403600)the National Natural Science Foundation of China(W2433030)the Key Research and Development Program of Jiangsu Province(BE2021691)。
文摘Laccases,belonging to the superfamily of multicopper oxidases,can perform electron oxidation on a broad range of substrates,releasing only water as a by-product.Although instability and aggregation significantly constrain the industrial use of these eco-friendly biocatalysts,it is a daunting challenge for current engineering strategies to elevate these crucial enzymatic characteristics simultaneously.Here,we developed a cyclizing laccase(CyLacc)using SpyTag/SpyCatcher technology,which endows the enzyme with high thermostability and high solubility.Differential scanning calorimetry sheds light on the thermal unfolding and refolding processes of CyLacc,indicating its increased tolerance to high-temperature environments.Furthermore,utilizing large-scale molecular dynamics simulations,we found that the reduction of the hydrophobic surface and the increased flexibility of the loops are the main reasons for the increase in solubility.Compared with the wild-type laccase,the improved features led to a much higher alkaline lignin,Malachite Green,and Neutral Red decolorization efficiency of CyLacc.Furthermore,CyLacc significantly enhanced the polymerization yields of catechol and hydroquinone,from 49%and 63.6%(with wild-type Lacc)to 78.5%and 90.3%,respectively.The number-average molecular weights of the polyphenols ranged from 1,000 to 1,200 D(corresponding to the degree of polymerization that varied from 10 to 13),showing selective polymerization of phenolic compounds catalyzed by laccase.The present work,combining theory with experiment,opens up a new avenue for industrially important laccase engineering and is expected to contribute to lignin valorization and environmental protection.
基金financial supports from the Research Grants Council of Hong Kong SAR Government to F. Sun (RGC-ECS Nos. #26103915 and Ao E/M-09/12)the 863 Program (No. 2015AA020941)+2 种基金the National Natural Science Foundation of China (Nos. 21474003, 91427304)"1000 Plan (Youth)"the Department of Chemical and Biological Engineering, HKUST for the faculty start-up fund
文摘Synthesis of macromolecular systems with precise structural and functional control constitutes a fundamental challenge for materials science and engineering. Development of the ability to construct complex bio-macromolecular architectures provides a solution to this challenge. The past few years have witnessed the emergence of a new category of peptide-protein chemistry which can covalently stitch together protein]peptide molecules with high specificity under mild physiological conditions. It has thus inspired the concept of genetically encoded click chemistry （GECC）. As a prototype of GECC, SpyTag/ SpyCatcher chemistry has enabled the precise synthesis ofmacromolecules both in vitro and in vivo, exerting precise control over the fundamental properties of these macromolecules including length, sequence, stereochemistry and topology and leading to the creation of diverse biomaterials for a variety of applications. We thus anticipate a potential toolbox of GECC comprising multiple mutually orthogonal, covalent-bond forming peptide-protein reactive pairs with diverse features, which shall bridge synthetic biology and materials science and open up enormous opportunities for biomaterialsin the future.
基金supported by the Zhejiang Foundation Public Welfare Research Project(Authorization No.:LGF19B060006)。
文摘Due to the complexity of bioactive ingredients in biological samples,the screening of target proteins is a complex process.Herein,a feasible strategy for directing protein immobilization on silica magnetic beads for ligand fishing based on SpyTag/SpyCatcher(ST/SC)-mediated anchoring is presented.Carboxyl functional groups on the surface of silica-coated magnetic beads(SMBs)were coupled with SC using the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride/N-hydroxysulfosuccinimide method,named SC-SMBs.The green fluorescent protein(GFP),as the capturing protein model,was ST-labeled and anchored at a specific orientation onto the surface of SC-SMBs directly from relevant cell lysates via ST/SC self-ligation.The characteristics of the SC-SMBs were studied via electron microscopy,energy dispersive spectroscopy,and Fourier transform infrared spectroscopy.The spontaneity and site-specificity of this unique reaction were confirmed via electrophoresis and fluorescence analyses.Although the alkaline stability of ST-GFP-ligated SC-SMBs was not ideal,the formed isopeptide bond was unbreakable under acidic conditions(0.05 M glycine-HCl buffer,pH 1e6)for 2 h,under 20%ethanol solution within 7 days,and at most temperatures.We,therefore,present a simple and universal strategy for the preparation of diverse protein-functionalized SMBs for ligand fishing,prompting its usage on drug screening and target finding.
基金supported by National Key Research and Develop-ment Program of China(2022YFC2104901)National Natural Science Foundation of China(32372279).
文摘Allitol is a valuable drug intermediate and a potential low-calorie sweetener in food industry.However,developing an efficient biotransformation system for large-scale production of allitol remains challenging.Here,a customized multienzyme cascade immobilization system was designed for efficient biocatalytic synthesis of allitol from D-fructose.The system included three immobilized enzymes:amino resin(LX1000)-immobilized Dallulose 3-epimerase(SfDAE)from Sinorhizobium fredii,LX1000-immobilized ribitol dehydrogenase(KpRDH)from Klebsiella pneumoniae,and derived formate dehydrogenase(CbFDH)from Candida boidinii was directional immobilization on bacterial cellulose(BC)employ SpyCatcher/SpyTag modular.By employing a strategy of directional immobilization and SpyCatcher-functionalized BC,the poor cascade cooperativity in the rate-limiting enzyme(CbFDH)was successfully resolved.Under the optimum biotransformation conditions,this system was able to convert 500 g/L of D-fructose into 455 g/L of allitol with conversion yields as high as 90%.Importantly,they also had excellent stability,retaining 60%relative activity after 10 repeated cycles,and preserving 50.2%relative activity after 24 days of storage.Consequently,the design and application of this multienzyme cascade immobilization system successfully provides an effective strategy for the large-scale biosynthesis of allitol.
