Production of economically viable bioethanol is potentially an environmentally and financially worthwhile endeavor.One major source for fermentable sugars is lignocellulose.However,lignocellulosic biomass is difficult...Production of economically viable bioethanol is potentially an environmentally and financially worthwhile endeavor.One major source for fermentable sugars is lignocellulose.However,lignocellulosic biomass is difficult to degrade,owing to its inherent structural recalcitrance.Cellulosomes are complexes of cellulases and associated polysaccharide-degrading enzymes bound to a protein scaffold that can efficiently degrade lignocellulose.Integration of the enzyme subunits into the complex depends on intermodular cohesin-dockerin interactions,which are robust but nonetheless non-covalent.The modular architecture of these complexes can be used to assemble artificial designer cellulosomes for advanced nanotechnological applications.Pretreatments that promote lignocellulose degradation involve high temperatures and acidic or alkaline conditions that could dismember designer cellulosomes,thus requiring separation of reaction steps,thereby increasing overall process cost.To overcome these challenges,we developed a means of covalently locking cohesin-dockerin interactions by integrating the chemistry of SpyCatcher-SpyTag approach to target and secure the interaction.The resultant cohesin-conjugated dockerin complex was resistant to high temperatures,SDS,and urea while high affinity and specificity of the interacting modular components were maintained.Using this approach,a covalently locked,bivalent designer cellulosome complex was produced and demonstrated to be enzymatically active on cellulosic substrates.The combination of affinity systems with SpyCatcher-SpyTag chemistry may prove of general use for improving other types of protein ligation systems and creating unconventional,biologically active,covalently locked,affinity-based molecular architectures.展开更多
Recent years have seen an ever increasing number of enzyme mediated protein/peptide modification reactions, which contribute significantly to the elucidation of related biological functions. The many available enzymes...Recent years have seen an ever increasing number of enzyme mediated protein/peptide modification reactions, which contribute significantly to the elucidation of related biological functions. The many available enzymes have, however, caused difficulties for practitioners in choosing the most appropriate enzyme for a certain purpose. This review surveyed the widely used enzymes(i.e., sortases, butelase 1,subtiligase, formylglycine generating enzyme and farnesyltransferase) in the manipulation of proteins/peptides, and the application fields of these enzymes as well as the advantages and limitations of each enzyme are summarized.展开更多
Solid phase peptide synthesis(SPPS)based on Fmoc chemistry has become a commonly used technique in peptide chemistry,as it can be easily conducted using automated machine,and not requiring highly toxic HF in compariso...Solid phase peptide synthesis(SPPS)based on Fmoc chemistry has become a commonly used technique in peptide chemistry,as it can be easily conducted using automated machine,and not requiring highly toxic HF in comparison to Boc-SPPS.With the fast development in the emerging field of protein chemical synthesis,many efforts have been endeavored aiming to find more efficient methods for preparing peptide fragments required in ligation reactions.This review briefly summarizes recent advances in the engineering and modification of Fmoc-SPPS-derived peptides,which can be used as the N-terminal fragments in a native chemical ligation(NCL)or NCL-type ligation reactions.展开更多
文摘Production of economically viable bioethanol is potentially an environmentally and financially worthwhile endeavor.One major source for fermentable sugars is lignocellulose.However,lignocellulosic biomass is difficult to degrade,owing to its inherent structural recalcitrance.Cellulosomes are complexes of cellulases and associated polysaccharide-degrading enzymes bound to a protein scaffold that can efficiently degrade lignocellulose.Integration of the enzyme subunits into the complex depends on intermodular cohesin-dockerin interactions,which are robust but nonetheless non-covalent.The modular architecture of these complexes can be used to assemble artificial designer cellulosomes for advanced nanotechnological applications.Pretreatments that promote lignocellulose degradation involve high temperatures and acidic or alkaline conditions that could dismember designer cellulosomes,thus requiring separation of reaction steps,thereby increasing overall process cost.To overcome these challenges,we developed a means of covalently locking cohesin-dockerin interactions by integrating the chemistry of SpyCatcher-SpyTag approach to target and secure the interaction.The resultant cohesin-conjugated dockerin complex was resistant to high temperatures,SDS,and urea while high affinity and specificity of the interacting modular components were maintained.Using this approach,a covalently locked,bivalent designer cellulosome complex was produced and demonstrated to be enzymatically active on cellulosic substrates.The combination of affinity systems with SpyCatcher-SpyTag chemistry may prove of general use for improving other types of protein ligation systems and creating unconventional,biologically active,covalently locked,affinity-based molecular architectures.
基金The financial support from the National Recruitment Program of Global Youth Experts(1000 Talents Plan)the National Natural Science Foundation of China (No. 81703406)
文摘Recent years have seen an ever increasing number of enzyme mediated protein/peptide modification reactions, which contribute significantly to the elucidation of related biological functions. The many available enzymes have, however, caused difficulties for practitioners in choosing the most appropriate enzyme for a certain purpose. This review surveyed the widely used enzymes(i.e., sortases, butelase 1,subtiligase, formylglycine generating enzyme and farnesyltransferase) in the manipulation of proteins/peptides, and the application fields of these enzymes as well as the advantages and limitations of each enzyme are summarized.
基金supported by the Peking University Health Science Center(BMU20130354)State Key Laboratory of Natural and Biomimetic Drugs,the National Recruitment Program of Global Youth Experts(1000 Plan)the National Natural Science Foundation of China (21502005)
文摘Solid phase peptide synthesis(SPPS)based on Fmoc chemistry has become a commonly used technique in peptide chemistry,as it can be easily conducted using automated machine,and not requiring highly toxic HF in comparison to Boc-SPPS.With the fast development in the emerging field of protein chemical synthesis,many efforts have been endeavored aiming to find more efficient methods for preparing peptide fragments required in ligation reactions.This review briefly summarizes recent advances in the engineering and modification of Fmoc-SPPS-derived peptides,which can be used as the N-terminal fragments in a native chemical ligation(NCL)or NCL-type ligation reactions.
