Protein thermostability is an inherent characteristic of proteins from thermophilic microorganisms,and therefore enables these organisms to survive at extreme temperatures.Although it is well-known that thermostable p...Protein thermostability is an inherent characteristic of proteins from thermophilic microorganisms,and therefore enables these organisms to survive at extreme temperatures.Although it is well-known that thermostable proteins are critical for the growth of thermophilic organisms,the structural basis of protein thermostability is not yet fully understood.The histidine-containing phosphocarrier (HPr) protein,a phosphate shuttle protein in the phosphoenolpyruvate-dependent sugar transport system (PTS) of bacterial species,is an ideal model for investigating protein thermostability with respect to its small size and deficiency in disulphide bonds or cofactors.In this study,the HPr protein from Thermoanaerobacter tengcongensis (TtHPr) is cloned and purified.Crystal structure with good quality has been determined at 2.3 resolution,which provides a firm foundation for exploring the thermostable mechanism.However,it shows that the crystal structure is conserved and no clue can be obtained from this single structure.Furthermore,detailed comparison of sequence and structure with the homologs from mesoor thermophilic bacteria shows no obvious rule for thermostability,but the extra salt-bridge existing only in thermophilic bacteria might be a better explanation for thermostability of HPr.Thus,mutations are performed to interrupt the salt-bridge in HPrs in thermophilic bacteria.Using site-directed mutations and the circular dichroism method,thermostability is evaluated,and the mutational variations are shown to have a faster denaturing rate than for wild-type viruses,indicating that mutations cause instability in the HPrs.Understanding the higher-temperature resistance of thermophilic and hyperthermophilic proteins is essential to studies on protein folding and stability,and is critical in engineering efficient enzymes that can work at a high temperature.展开更多
To determine the impact of carbohydrates on the metabolic pathway in alkaliphiles, proteomes were obtained from cultures containing different carbohydrates and were resolved on two-dimensional gel electrophoresis (2-D...To determine the impact of carbohydrates on the metabolic pathway in alkaliphiles, proteomes were obtained from cultures containing different carbohydrates and were resolved on two-dimensional gel electrophoresis (2-DE). The proteomes were compared to determine differentially expressed proteins. A novel alkaliphilic bacterium (alkaliphilic Bacillus sp. N16-5 isolated from Wudunur Soda Lake, China) was isolated in media with five different carbon sources (glucose, mannose, galactose, arabinose, and xylose). Comparative proteome analysis identified 61 differentially expressed proteins, which were mainly involved in carbohydrate metabolism, amino acid transport, and metabolism, as well as energy production and conversion. The comparison was based on the draft genome sequence of strain N16-5. The abundance of enzymes involved in central metabolism was significantly changed when exposed to various carbohydrates. Notably, catabolite control protein A (CcpA) was up-regulated under all carbon sources compared with glucose. In addition, pentose exhibited a stronger effect than hexose in CcpA-mediated carbon catabolite repression. These results provided a fundamental understanding of carbohydrate metabolism in alkaliphiles.展开更多
Chitin is the second most abundant renewable biopolymer in the world.Chitinases play important roles in the degradation of chitin.Chitinases are produced by different organisms for different purposes,which are widely ...Chitin is the second most abundant renewable biopolymer in the world.Chitinases play important roles in the degradation of chitin.Chitinases are produced by different organisms for different purposes,which are widely expressed in the three domains of life,ranging from archaea,bacteria,to fungi,yeasts,plants,insects,and even vertebrates.But there are few reports about Saccharomyces cerevisiae chitinase(ScCTS1).The aim of this study was to realize the high level expression of ScCTS1.The ScCTS1 was cloned into the expression vector pPIC9K.The recombinant plasmid was linearized and transformed into competent Pichia pastoris GS115.After screening by G418 plate,the fermentation conditions were optimized.Ultimately,under the optimal fermentation conditions,ScCTS1 enzymatic activity reached up to 94.6 U/mL.This paper presents the first report on the heterologous expression of a full-length ScCTS1 with considerably high activity.The work will not only make a great stride towards its potential applications in biotechnology,but also facilitate elucidating the precise mechanism of yeast cell division.展开更多
基金supported by the National High Technology Research and Development Program of China (Grant No. 2006BAD06A04) from Ministry of Science and Technology of ChinaGeorge F. Gao is a leading principal investigator of the Innovative Research Group of the National Natural Science Foundation of China (Grant No. 80121003)
文摘Protein thermostability is an inherent characteristic of proteins from thermophilic microorganisms,and therefore enables these organisms to survive at extreme temperatures.Although it is well-known that thermostable proteins are critical for the growth of thermophilic organisms,the structural basis of protein thermostability is not yet fully understood.The histidine-containing phosphocarrier (HPr) protein,a phosphate shuttle protein in the phosphoenolpyruvate-dependent sugar transport system (PTS) of bacterial species,is an ideal model for investigating protein thermostability with respect to its small size and deficiency in disulphide bonds or cofactors.In this study,the HPr protein from Thermoanaerobacter tengcongensis (TtHPr) is cloned and purified.Crystal structure with good quality has been determined at 2.3 resolution,which provides a firm foundation for exploring the thermostable mechanism.However,it shows that the crystal structure is conserved and no clue can be obtained from this single structure.Furthermore,detailed comparison of sequence and structure with the homologs from mesoor thermophilic bacteria shows no obvious rule for thermostability,but the extra salt-bridge existing only in thermophilic bacteria might be a better explanation for thermostability of HPr.Thus,mutations are performed to interrupt the salt-bridge in HPrs in thermophilic bacteria.Using site-directed mutations and the circular dichroism method,thermostability is evaluated,and the mutational variations are shown to have a faster denaturing rate than for wild-type viruses,indicating that mutations cause instability in the HPrs.Understanding the higher-temperature resistance of thermophilic and hyperthermophilic proteins is essential to studies on protein folding and stability,and is critical in engineering efficient enzymes that can work at a high temperature.
基金supported by the National Basic Research Program of China, Ministry of Science and Technology of China (Grant Nos. 2007CB707801 and 2003CB716001)the National High Technology Research and Development Program of China (Grant Nos. 2006AA020201 and 2007AA021306)
文摘To determine the impact of carbohydrates on the metabolic pathway in alkaliphiles, proteomes were obtained from cultures containing different carbohydrates and were resolved on two-dimensional gel electrophoresis (2-DE). The proteomes were compared to determine differentially expressed proteins. A novel alkaliphilic bacterium (alkaliphilic Bacillus sp. N16-5 isolated from Wudunur Soda Lake, China) was isolated in media with five different carbon sources (glucose, mannose, galactose, arabinose, and xylose). Comparative proteome analysis identified 61 differentially expressed proteins, which were mainly involved in carbohydrate metabolism, amino acid transport, and metabolism, as well as energy production and conversion. The comparison was based on the draft genome sequence of strain N16-5. The abundance of enzymes involved in central metabolism was significantly changed when exposed to various carbohydrates. Notably, catabolite control protein A (CcpA) was up-regulated under all carbon sources compared with glucose. In addition, pentose exhibited a stronger effect than hexose in CcpA-mediated carbon catabolite repression. These results provided a fundamental understanding of carbohydrate metabolism in alkaliphiles.
基金This work was supported by the General Project of Beijing Municipal Education Commission(No.SQKM201311417003)Beijing Excellent Talents Cultivation Project(No.2012D005022000007)Ministry of Science and Technology“863 Plan”Project(No.2015AA020202).
文摘Chitin is the second most abundant renewable biopolymer in the world.Chitinases play important roles in the degradation of chitin.Chitinases are produced by different organisms for different purposes,which are widely expressed in the three domains of life,ranging from archaea,bacteria,to fungi,yeasts,plants,insects,and even vertebrates.But there are few reports about Saccharomyces cerevisiae chitinase(ScCTS1).The aim of this study was to realize the high level expression of ScCTS1.The ScCTS1 was cloned into the expression vector pPIC9K.The recombinant plasmid was linearized and transformed into competent Pichia pastoris GS115.After screening by G418 plate,the fermentation conditions were optimized.Ultimately,under the optimal fermentation conditions,ScCTS1 enzymatic activity reached up to 94.6 U/mL.This paper presents the first report on the heterologous expression of a full-length ScCTS1 with considerably high activity.The work will not only make a great stride towards its potential applications in biotechnology,but also facilitate elucidating the precise mechanism of yeast cell division.
