Archaea represents the third domain of life, with the information-processing machineries more closely resembling those of eukaryotes than the machineries of the bacterial counterparts but sharing metabolic pathways wi...Archaea represents the third domain of life, with the information-processing machineries more closely resembling those of eukaryotes than the machineries of the bacterial counterparts but sharing metabolic pathways with organisms of Bacteria, the sister prokaryotic phylum. Archaeal organisms also possess unique features as revealed by genomics and genome comparisons and by biochemical characterization of prominent enzymes. Nevertheless, diverse genetic tools are required for in vivo experiments to verify these interesting discoveries. Considerable efforts have been devoted to the development of genetic tools for archaea ever since their discovery, and great progress has been made in the creation of archaeal genetic tools in the past decade. Versatile genetic toolboxes are now available for several archaeal models, among which Sulfolobus microorganisms are the only genus representing Crenarchaeota because all the remaining genera are from Euryarchaeota. Nevertheless, genetic tools developed for Sulfolobus are probably the most versatile among all archaeal models, and these include viral and plasmid shuttle vectors, conventional and novel genetic manipulation methods, CRISPR-based gene deletion and mutagenesis, and gene silencing, among which CRISPR tools have been reported only for Sulfolobus thus far. In this review, we summarize recent developments in all these useful genetic tools and discuss their possible application to research into archaeal biology by means of Sulfolobus models.展开更多
RecA family recombinases play essential roles in maintaining genome integrity. A group of RecA-like proteins named RadC are present in all archaea, but their in vivo functions remain unclear. In this study, we perform...RecA family recombinases play essential roles in maintaining genome integrity. A group of RecA-like proteins named RadC are present in all archaea, but their in vivo functions remain unclear. In this study, we performed phylogenetic and genetic analysis of two RadC proteins from Sulfolobus islandicus. RadC is closer to the KaiC lineage of cyanobacteria and proteobacteria than to the lineage of the recombinases （RecA, RadA, and Rad51） and the recombinase paralogs （e.g., RadB, Rad55, and Rad51B）. Using the recently- established S. islandicus genetic system, we constructed deletion and over-expression strains of radC1 and radC2. Deletion of radC1 rendered the cells more sensitive to DNA damaging agents, methyl methanesulfonate （MMS）, hydroxyurea （HU）, and ultraviolet （UV） radiation, than the wild type, and a AradCIAradC2 double deletion strain was more sensitive to cisplatin and MMS than the AradC1 single deletion mutant. In addition, ectopic expression of His-tagged RadC 1 revealed that RadC I was co-purified with a putative structure-specific nuclease and ATPase, which is highly conserved in archaea. Our results indicate that both RadCI and RadC2 are involved in DNA repair. RadCl may play a general or primary role in DNA repair, while RadC2 plays a role in DNA repair in response to specific DNA damages.展开更多
To study the DNA synthesis mechanism of Sulfolobus acidocaldarius, a thermophilic species from Crenarehaeota, two DNA polymerases of B family（polB1 and polB3）, and one DNA polymerase of Y family（polIV） were recomb...To study the DNA synthesis mechanism of Sulfolobus acidocaldarius, a thermophilic species from Crenarehaeota, two DNA polymerases of B family（polB1 and polB3）, and one DNA polymerase of Y family（polIV） were recombinantly expressed, purified and biochemically characterized. Both DNA polymerases polBl（Saci_1537） and polB3（Saci_0074） possessed DNA polymerase and 3＇ to 5＇ exonuclease activities; however, both the activities of B3 were very inefficient in vitro. The polIV（Saci_0554） was a polymerase, not an exonuclease. The activities of all the three DNA polymerases were dependent on divalent metal ions Mn1＋ and Mg2＋. They showed the highest activity at pH values ranging from 8.0 to 9.5. Their activities were inhibited by KC1 with high concentration. The optimal reaction temperatures for the three DNA polymerases were between 60 and 70 ℃. Deaminated bases dU and dI on DNA template strongly hindered primer extension by the two DNA polymerases of B family, not by the DNA polymerase of Y family. DNA polymerase of Y Family bypassed the two AP site analogues dSpacer and propane on template more easily than DNA polymerases of B family. Our results suggest that the three DNA polymerases coordinate to fulfill various DNA synthesis in Sulfolobus acidocaldarius cell.展开更多
Sulfur speciation transformation during bioleaching of pyrite-containing sphalerite concentrate by thermophile Sulfolobus metallicus （S. metallicus） at 65 ℃ was investigated by X-ray diffraction （XRD）, diffuse re...Sulfur speciation transformation during bioleaching of pyrite-containing sphalerite concentrate by thermophile Sulfolobus metallicus （S. metallicus） at 65 ℃ was investigated by X-ray diffraction （XRD）, diffuse reflectance Fourier transform infrared spectroscopy （FT-IR） and sulfur K-edge X-ray absorption near edge structure spectroscopy （XANES）. The results show that the presence of S. metallicus effectively enhances the dissolution of the mineral. The yield of zinc increases from 0.5 g/L in sterile control to 2.7 g/L in bioleaching. The pyrite in the concentrate facilitates zinc dissolution in the early stage, but has hindrance role in the late stage for the formation of jarosite. Sulfur speciation analyses show that jarosite and elemental sulfur are main products in bioleaching process, and the accumulation ofjarosite is mainly responsible for the decline of leaching efficiency.展开更多
In archaea, the HEL308 homolog Hel308a（or Hjm） is implicated in stalled replication fork repair. The biochemical properties and structures of Hjm homologs are well documented, but in vivo mechanistic information is ...In archaea, the HEL308 homolog Hel308a（or Hjm） is implicated in stalled replication fork repair. The biochemical properties and structures of Hjm homologs are well documented, but in vivo mechanistic information is limited. Herein, a structure-based functional analysis of Hjm was performed in the genetically tractable hyperthermophilic archaeon, Sulfolobus islandicus. Results showed that domain V and residues within it, which affect Hjm activity and regulation, are essential and that the domain V-truncated mutants and sitedirected mutants within domain V cannot complement hjm chromosomal deletion. Chromosomal hjm deletion can be complemented by ectopic expression of hjm under the control of its native promoter but not an artificial arabinose promoter. Cellular Hjm levels are kept constant under ultraviolet（UV） and methyl methanesulfonate（MMS） treatment conditions in a strain carrying a plasmid to induce Hjm overexpression. These results suggest that Hjm expression and activity are tightly controlled, probably at the translational level.展开更多
The chalcopyrite-adsorption characteristics and leaching properties of Sulfolobus metallicus（S. metallicus） YN24 were investigated in this study. The effects of zeta potentials of S. metallicus samples on chalcopyri...The chalcopyrite-adsorption characteristics and leaching properties of Sulfolobus metallicus（S. metallicus） YN24 were investigated in this study. The effects of zeta potentials of S. metallicus samples on chalcopyrite cultivated with distinct sources of energy were similar. Regardless of the energy source cultivated, all of the investigated S. metallicus samples adhered rapidly to the chalcopyrite surface, with an adhesion plateau being reached within 60 min. However, the mineral-cultured S. metallicus adsorbed more strongly onto chalcopyrite than the sulfur-cultured S. metallicus did. Furthermore, chalcopyrite-leaching tests suggested that the copper-leaching ability of the mineral-cultured S. metallicus was also greater than that of unadapted S. metallicus. Therefore, the results provide insights into the mechanism of mineral-surface adsorption of microorganisms that helps enhance the copper-leaching rate.展开更多
Topoisomerase III （topo III）, a type IA topoisomerase, is widespread in hyperthermophilic archaea. In order to interrogate the in vivo role of archaeal topo III, we constructed and characterized a topo III gene dele...Topoisomerase III （topo III）, a type IA topoisomerase, is widespread in hyperthermophilic archaea. In order to interrogate the in vivo role of archaeal topo III, we constructed and characterized a topo III gene deletion mutant of Sulfolobus islandicus. The mutant was ,viable but grew more slowly than the wild-type strain, especially in a nutrient-poor medium. Flow cytometry analysis revealed changes of the mutant in growth cycle characteristics including an increase in proportion of cells containing either more than two genome equivalents or less than one genome equivalent in exponentially-growing cultures. As shown by fluorescence microscopy, a fraction of mutant cells in the cultures were drastically enlarged, and at least some of the enlarged cells were apparently capable of resuming cell division. The mutant also shows a different tran- scriptional profile from that of the wild-type strain. Our results suggest that the enzyme may serve roles in chromosomal segregation and control of the level of supercoiling in the cell.展开更多
An 11.5-ku DNA binding protein, designated as Sshl2, was purified from the hyperthermophilic archaeon Sulfolobus shibatae by column chromatography in SP Sepharose, DNA cellulose and phosphocellulose. Sshl2 accounts fo...An 11.5-ku DNA binding protein, designated as Sshl2, was purified from the hyperthermophilic archaeon Sulfolobus shibatae by column chromatography in SP Sepharose, DNA cellulose and phosphocellulose. Sshl2 accounts for about 4 % of the total cellular protein. The protein is capable of binding to both negatively supercoiled and relaxed DNAs. Nick closure analysis revealed that Sshl2 constrains negative supercoils upon binding to DNA. While the ability of the protein to constrain supercoils is weak at 22℃ , it is enhanced substantially at temperatures higher than 37℃ . Both the cellular content and supercoil-constraining ability of Sshl2 suggest that the protein may play an important role in the organization and stabilization of the chromosome of S. shibatae.展开更多
Rad51/RadA paralogs found in eukaryotes and euryarchaea play important roles during recombination and repair,and mutations in one of the human Rad51 paralogs,Rad51C,are associated with breast and ovarian cancers.The h...Rad51/RadA paralogs found in eukaryotes and euryarchaea play important roles during recombination and repair,and mutations in one of the human Rad51 paralogs,Rad51C,are associated with breast and ovarian cancers.The hyperthermophilic crenarchaeon Sulfolobus tokodaii encodes four putative RadA paralogs and studies on these proteins may assist in understanding the functions of human Rad51 paralogs.Here,we report the biochemical characterization of stRadC2,a S.tokodaii RadA paralog.Pull-down assays revealed that the protein was able to interact with the recombinase,RadA,and the Holliday junction endonuclease,Hjc.stRadC2 inhibited the strand exchange activity of RadA and facilitated Hjc-mediated Holliday junction DNA cleavage in vitro.RT-PCR analysis revealed that stRadC2 transcription was immediately reduced after UV irradiation,but was restored to normal levels at the late stages of DNA repair.Our results suggest that stRadC2 may act as an anti-recombination factor in DNA recombinational repair in S.tokodaii.展开更多
The temperate virus SSV1 from the hyper-thermophilic archaeon Sulfolobus shibatae provides a useful model system for the study of archaeal DNA replication. Southern hybridization showed that SSV1 existed primarily as ...The temperate virus SSV1 from the hyper-thermophilic archaeon Sulfolobus shibatae provides a useful model system for the study of archaeal DNA replication. Southern hybridization showed that SSV1 existed primarily as a provirus in its host that was grown without shaking. Upon UV or mitomycin C induction, the cellular level of free SSV1 DNA increased drastically whereas that of integrated viral DNA remained unchanged. The results of mitomycin C induction were more reproducible than those of UV induction. We found that, when the cells that had been grown without shaking were shaken, the replication of SSV1 DNA was also induced. Based on our results, we developed a method for the induction of SSY1 DNA replication by mitomycin C. When the S. shibatae virus production was induced using this method, the cellular level of free SSV1 DNA started to increase 10 h after induction, and peaked after 12-15 h. A fully induced S. shibatae cell contained -50 molecules of free SSV1 DNA. The development of this induction展开更多
Acyl-peptide releasing enzyme（AARE） belongs to a serine peptidase family and catalyzes the NH2-terminal hydrolysis of Nα-acylpeptides to release Nα-acylated amino acids. ORF0779（ORF=open reading frame） from ther...Acyl-peptide releasing enzyme（AARE） belongs to a serine peptidase family and catalyzes the NH2-terminal hydrolysis of Nα-acylpeptides to release Nα-acylated amino acids. ORF0779（ORF=open reading frame） from thermophilic archaea Sulfolobus tokodaii（ST0779） was cloned and expressed in E. coli BL21 and the expressed protein was identified as a thermostable AARE. The target protein could be optimally overexpressed in E. coli at 30 °C for 8 h with 0.1 mmol/L isopropyl β-dthiogalactoside（IPTG）. The crude enzyme was heated at 70 °C for 30 min, and then the target protein could account for above 40% of the total protein. The purification fold was 27 and the enzyme showed both esterase activity and peptidase activity. The optimal temperature and pH for ST0779 were 70 °C and 8.0 when Ac-Ala3 was used as substrate. The half-life of the enzyme（0.2 mg/mL） at 90 °C was about 16 h, indicating that the enzyme exhibits a favorable thermostability. The activity of ST0779 could still remain over 85% after being treated at 25 °C in different buffers with pH range from 6.0 to10.0 for 24 h, which indicates ST0779 is stable in neutral or slight alkali environment. Under neutral or slightly alkali conditions, the enzyme exhibits really high catalytic efficiency against acyl-peptide, and the optimal substrate is Ac-Ala3. Most metal ions have no inhibition effect on the activity of ST0779, while 4% activity of ST0779 is inhibited in the presence of K＋. This enzyme was supposed to be applied in the analysis of protein sequencing and the synthesis of small peptides.展开更多
基金supported by the Danish Council of Independent Research (DFF-0602-02196, DFF-4181-00274, DFF-1323-00330)the Fundamental Research Funds for the Central Universities (2662015PX199)
文摘Archaea represents the third domain of life, with the information-processing machineries more closely resembling those of eukaryotes than the machineries of the bacterial counterparts but sharing metabolic pathways with organisms of Bacteria, the sister prokaryotic phylum. Archaeal organisms also possess unique features as revealed by genomics and genome comparisons and by biochemical characterization of prominent enzymes. Nevertheless, diverse genetic tools are required for in vivo experiments to verify these interesting discoveries. Considerable efforts have been devoted to the development of genetic tools for archaea ever since their discovery, and great progress has been made in the creation of archaeal genetic tools in the past decade. Versatile genetic toolboxes are now available for several archaeal models, among which Sulfolobus microorganisms are the only genus representing Crenarchaeota because all the remaining genera are from Euryarchaeota. Nevertheless, genetic tools developed for Sulfolobus are probably the most versatile among all archaeal models, and these include viral and plasmid shuttle vectors, conventional and novel genetic manipulation methods, CRISPR-based gene deletion and mutagenesis, and gene silencing, among which CRISPR tools have been reported only for Sulfolobus thus far. In this review, we summarize recent developments in all these useful genetic tools and discuss their possible application to research into archaeal biology by means of Sulfolobus models.
基金supported by the grants from the National Natural Science Foundation of China(Nos.3093002 and 31170072) to Y.SDanish Council of Independent Research(No.FTP/11-106683) to Q.S
文摘RecA family recombinases play essential roles in maintaining genome integrity. A group of RecA-like proteins named RadC are present in all archaea, but their in vivo functions remain unclear. In this study, we performed phylogenetic and genetic analysis of two RadC proteins from Sulfolobus islandicus. RadC is closer to the KaiC lineage of cyanobacteria and proteobacteria than to the lineage of the recombinases （RecA, RadA, and Rad51） and the recombinase paralogs （e.g., RadB, Rad55, and Rad51B）. Using the recently- established S. islandicus genetic system, we constructed deletion and over-expression strains of radC1 and radC2. Deletion of radC1 rendered the cells more sensitive to DNA damaging agents, methyl methanesulfonate （MMS）, hydroxyurea （HU）, and ultraviolet （UV） radiation, than the wild type, and a AradCIAradC2 double deletion strain was more sensitive to cisplatin and MMS than the AradC1 single deletion mutant. In addition, ectopic expression of His-tagged RadC 1 revealed that RadC I was co-purified with a putative structure-specific nuclease and ATPase, which is highly conserved in archaea. Our results indicate that both RadCI and RadC2 are involved in DNA repair. RadCl may play a general or primary role in DNA repair, while RadC2 plays a role in DNA repair in response to specific DNA damages.
基金Supported by the National Natural Science Foundation of China（No.31371260） and the Natural Science Foundation of Shanghai City, China（No. 12ZR1413700）. We greatly appreciate Professor Sonja-Verena Albers for kindly providing the strain of S. acidocaldarius.
文摘To study the DNA synthesis mechanism of Sulfolobus acidocaldarius, a thermophilic species from Crenarehaeota, two DNA polymerases of B family（polB1 and polB3）, and one DNA polymerase of Y family（polIV） were recombinantly expressed, purified and biochemically characterized. Both DNA polymerases polBl（Saci_1537） and polB3（Saci_0074） possessed DNA polymerase and 3＇ to 5＇ exonuclease activities; however, both the activities of B3 were very inefficient in vitro. The polIV（Saci_0554） was a polymerase, not an exonuclease. The activities of all the three DNA polymerases were dependent on divalent metal ions Mn1＋ and Mg2＋. They showed the highest activity at pH values ranging from 8.0 to 9.5. Their activities were inhibited by KC1 with high concentration. The optimal reaction temperatures for the three DNA polymerases were between 60 and 70 ℃. Deaminated bases dU and dI on DNA template strongly hindered primer extension by the two DNA polymerases of B family, not by the DNA polymerase of Y family. DNA polymerase of Y Family bypassed the two AP site analogues dSpacer and propane on template more easily than DNA polymerases of B family. Our results suggest that the three DNA polymerases coordinate to fulfill various DNA synthesis in Sulfolobus acidocaldarius cell.
基金Project(50974140) supported by the National Natural Science Foundation of ChinaProject(VR-09157) supported by Beijing Synchrotron Radiation Facility (BSRF) Public User Program,China
文摘Sulfur speciation transformation during bioleaching of pyrite-containing sphalerite concentrate by thermophile Sulfolobus metallicus （S. metallicus） at 65 ℃ was investigated by X-ray diffraction （XRD）, diffuse reflectance Fourier transform infrared spectroscopy （FT-IR） and sulfur K-edge X-ray absorption near edge structure spectroscopy （XANES）. The results show that the presence of S. metallicus effectively enhances the dissolution of the mineral. The yield of zinc increases from 0.5 g/L in sterile control to 2.7 g/L in bioleaching. The pyrite in the concentrate facilitates zinc dissolution in the early stage, but has hindrance role in the late stage for the formation of jarosite. Sulfur speciation analyses show that jarosite and elemental sulfur are main products in bioleaching process, and the accumulation ofjarosite is mainly responsible for the decline of leaching efficiency.
基金supported by the grants from the National Natural Science Foundation of China (Nos. 3093002, 31170072 and 31470184 to Y.S.)
文摘In archaea, the HEL308 homolog Hel308a（or Hjm） is implicated in stalled replication fork repair. The biochemical properties and structures of Hjm homologs are well documented, but in vivo mechanistic information is limited. Herein, a structure-based functional analysis of Hjm was performed in the genetically tractable hyperthermophilic archaeon, Sulfolobus islandicus. Results showed that domain V and residues within it, which affect Hjm activity and regulation, are essential and that the domain V-truncated mutants and sitedirected mutants within domain V cannot complement hjm chromosomal deletion. Chromosomal hjm deletion can be complemented by ectopic expression of hjm under the control of its native promoter but not an artificial arabinose promoter. Cellular Hjm levels are kept constant under ultraviolet（UV） and methyl methanesulfonate（MMS） treatment conditions in a strain carrying a plasmid to induce Hjm overexpression. These results suggest that Hjm expression and activity are tightly controlled, probably at the translational level.
基金financially supported by the National Basic Research Priorities Program of China (No. 2010CB630905)the National High-Tech Research and Development Program of China (No. 2012AA061501)
文摘The chalcopyrite-adsorption characteristics and leaching properties of Sulfolobus metallicus（S. metallicus） YN24 were investigated in this study. The effects of zeta potentials of S. metallicus samples on chalcopyrite cultivated with distinct sources of energy were similar. Regardless of the energy source cultivated, all of the investigated S. metallicus samples adhered rapidly to the chalcopyrite surface, with an adhesion plateau being reached within 60 min. However, the mineral-cultured S. metallicus adsorbed more strongly onto chalcopyrite than the sulfur-cultured S. metallicus did. Furthermore, chalcopyrite-leaching tests suggested that the copper-leaching ability of the mineral-cultured S. metallicus was also greater than that of unadapted S. metallicus. Therefore, the results provide insights into the mechanism of mineral-surface adsorption of microorganisms that helps enhance the copper-leaching rate.
基金supported by the National Natural Science Foundation of China(Nos.30921065,30730003 and 30870058) to L.Huangthe Danish Research Council for Technology and Production(No.274-07-0116)
文摘Topoisomerase III （topo III）, a type IA topoisomerase, is widespread in hyperthermophilic archaea. In order to interrogate the in vivo role of archaeal topo III, we constructed and characterized a topo III gene deletion mutant of Sulfolobus islandicus. The mutant was ,viable but grew more slowly than the wild-type strain, especially in a nutrient-poor medium. Flow cytometry analysis revealed changes of the mutant in growth cycle characteristics including an increase in proportion of cells containing either more than two genome equivalents or less than one genome equivalent in exponentially-growing cultures. As shown by fluorescence microscopy, a fraction of mutant cells in the cultures were drastically enlarged, and at least some of the enlarged cells were apparently capable of resuming cell division. The mutant also shows a different tran- scriptional profile from that of the wild-type strain. Our results suggest that the enzyme may serve roles in chromosomal segregation and control of the level of supercoiling in the cell.
基金Project supported by the National Natural Science Foundation of China (Grant No. 39770006)the Irvine Foundation and the Director's fund in the Institute of Microbiologythe Chinese Academy of Sciences, and a Grant-in-Aid for Scientists Returning f
文摘An 11.5-ku DNA binding protein, designated as Sshl2, was purified from the hyperthermophilic archaeon Sulfolobus shibatae by column chromatography in SP Sepharose, DNA cellulose and phosphocellulose. Sshl2 accounts for about 4 % of the total cellular protein. The protein is capable of binding to both negatively supercoiled and relaxed DNAs. Nick closure analysis revealed that Sshl2 constrains negative supercoils upon binding to DNA. While the ability of the protein to constrain supercoils is weak at 22℃ , it is enhanced substantially at temperatures higher than 37℃ . Both the cellular content and supercoil-constraining ability of Sshl2 suggest that the protein may play an important role in the organization and stabilization of the chromosome of S. shibatae.
基金supported by the National Natural Science Foundation of China (Grant Nos. 3093002 and 30870046 to Shen YuLong,and 30700011 to Sheng DuoHong)the Promotive Research Fund for Excellent Young and Middle-aged Scientists of Shandong Province (Grant No. BS2010SW014 to Sheng DuoHong)
文摘Rad51/RadA paralogs found in eukaryotes and euryarchaea play important roles during recombination and repair,and mutations in one of the human Rad51 paralogs,Rad51C,are associated with breast and ovarian cancers.The hyperthermophilic crenarchaeon Sulfolobus tokodaii encodes four putative RadA paralogs and studies on these proteins may assist in understanding the functions of human Rad51 paralogs.Here,we report the biochemical characterization of stRadC2,a S.tokodaii RadA paralog.Pull-down assays revealed that the protein was able to interact with the recombinase,RadA,and the Holliday junction endonuclease,Hjc.stRadC2 inhibited the strand exchange activity of RadA and facilitated Hjc-mediated Holliday junction DNA cleavage in vitro.RT-PCR analysis revealed that stRadC2 transcription was immediately reduced after UV irradiation,but was restored to normal levels at the late stages of DNA repair.Our results suggest that stRadC2 may act as an anti-recombination factor in DNA recombinational repair in S.tokodaii.
基金This work was supported by the National Natural Science Foundation of China (Grant Nos. 39770009 and 39925001)the fund for Knowledge Innovation Program of the Chinese Academy of Sciences (Grant No. KSCX2-3-01-02).
文摘The temperate virus SSV1 from the hyper-thermophilic archaeon Sulfolobus shibatae provides a useful model system for the study of archaeal DNA replication. Southern hybridization showed that SSV1 existed primarily as a provirus in its host that was grown without shaking. Upon UV or mitomycin C induction, the cellular level of free SSV1 DNA increased drastically whereas that of integrated viral DNA remained unchanged. The results of mitomycin C induction were more reproducible than those of UV induction. We found that, when the cells that had been grown without shaking were shaken, the replication of SSV1 DNA was also induced. Based on our results, we developed a method for the induction of SSY1 DNA replication by mitomycin C. When the S. shibatae virus production was induced using this method, the cellular level of free SSV1 DNA started to increase 10 h after induction, and peaked after 12-15 h. A fully induced S. shibatae cell contained -50 molecules of free SSV1 DNA. The development of this induction
基金Supported by the National Natural Science Foundation of China(Nos.20772046, 21072075)
文摘Acyl-peptide releasing enzyme（AARE） belongs to a serine peptidase family and catalyzes the NH2-terminal hydrolysis of Nα-acylpeptides to release Nα-acylated amino acids. ORF0779（ORF=open reading frame） from thermophilic archaea Sulfolobus tokodaii（ST0779） was cloned and expressed in E. coli BL21 and the expressed protein was identified as a thermostable AARE. The target protein could be optimally overexpressed in E. coli at 30 °C for 8 h with 0.1 mmol/L isopropyl β-dthiogalactoside（IPTG）. The crude enzyme was heated at 70 °C for 30 min, and then the target protein could account for above 40% of the total protein. The purification fold was 27 and the enzyme showed both esterase activity and peptidase activity. The optimal temperature and pH for ST0779 were 70 °C and 8.0 when Ac-Ala3 was used as substrate. The half-life of the enzyme（0.2 mg/mL） at 90 °C was about 16 h, indicating that the enzyme exhibits a favorable thermostability. The activity of ST0779 could still remain over 85% after being treated at 25 °C in different buffers with pH range from 6.0 to10.0 for 24 h, which indicates ST0779 is stable in neutral or slight alkali environment. Under neutral or slightly alkali conditions, the enzyme exhibits really high catalytic efficiency against acyl-peptide, and the optimal substrate is Ac-Ala3. Most metal ions have no inhibition effect on the activity of ST0779, while 4% activity of ST0779 is inhibited in the presence of K＋. This enzyme was supposed to be applied in the analysis of protein sequencing and the synthesis of small peptides.
