OBJECTIVETo clone the gene encoding adenylate kinase of Thermus thermophilus HB27, an extremely thermophilic bacterium, express the protein in Escherichia coil and study the enzymatic characterization.

METHODSThe DNA fragment encoding adenylate kinase was obtained by PCR from the total DNA of Thermus thermophilus HB27 and cloned into the vector pET-28a. The recombinant plasmid was identified by PCR, restriction endonuclease digestion and sequence analysis. Enzymatic characterization of the expressed protein was carried out using spectrophotometric assays.

RESULTSThe gene coding for adenylate kinase from Thermus thermophilus HB27 was cloned and the protein was overexpressed in Escherichia coli BL21(DE3). The optimum reactive pH and temperature for the enzyme were 8.5 and 90 degrees celsius;, respectively. The Km of the recombinant adenylate kinase for ADP was 68.6 micromol/L, with an V(max)ADP of 0.294 mmol/(L.min). Under the condition of environmental temperature at 70, 80, 90, or 100 degrees celsius; for 7 h, the recombinant adenylate kinase still retained the enzymatic activity with high thermostability. AP5A, a specific adenylate kinase inhibitor, inhibited the enzymatic activity of the protein by 70% at the concentration of 2.0 mmol/L, with a Ki value of 46.39 micromol/L for ADP.

CONCLUSIONThe gene coding for adenylate kinase of Thermus thermophilus HB27 has been successfully cloned and expressed in Escherichia coil, which provides the basis for potential use of the highly thermostable recombinant HB27 adenylate kinase.

Adenylate Kinase ; biosynthesis ; genetics ; metabolism ; Amino Acid Sequence ; Cloning, Molecular ; Enzyme Stability ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism ; Thermus thermophilus ; enzymology

Ribosomal RNAs are important because they catalyze the synthesis of peptides and proteins. Comparative studies of the secondary structure of 18S rRNA have revealed the basic locations of its many length-conserved and length-variable regions. In recent years, many more sequences of 18S rDNA with unusual lengths have been documented in GenBank. These data make it possible to recognize the diversity of the secondary and tertiary structures of 18S rRNAs and to identify the length-conserved parts of 18S rDNAs. The longest 18S rDNA sequences of almost every known eukaryotic phylum were included in this study. We illustrated the bioinformatics-based structure to show that, the regions that are more length-variable, regions that are less length-variable, the splicing sites for introns, and the sites of A-minor interactions are mostly distributed in different parts of the 18S rRNA. Additionally, this study revealed that some length-variable regions or insertion positions could be quite close to the functional part of the 18S rRNA of Foraminifera organisms. The tertiary structure as well as the secondary structure of 18S rRNA can be more diverse than what was previously supposed. Besides revealing how this interesting gene evolves, it can help to remove ambiguity from the alignment of eukaryotic 18S rDNAs and to improve the performance of 18S rDNA in phylogenetic reconstruction. Six nucleotides shared by Archaea and Eukaryota but rarely by Bacteria are also reported here for the first time, which might further support the supposed origin of eukaryote from archaeans.
Animals ; Base Sequence ; Drosophila melanogaster ; genetics ; Eukaryota ; classification ; Molecular Sequence Data ; Nucleic Acid Conformation ; Phylogeny ; RNA, Ribosomal, 16S ; chemistry ; genetics ; RNA, Ribosomal, 18S ; chemistry ; classification ; genetics ; Sequence Alignment ; Sequence Analysis, RNA ; Thermus thermophilus ; genetics

To obtain thermostable aspartate aminotransferase, the gene aspC from an extremely thermophilic bacterium, Thermus thermophilus HB8 was cloned, and its product was overexpressed in Escherichia coli BL21 (DE3) and Rosetta (DE3). The expression in Rosetta (DP3) was more efficient. The optimum reactive pH was 7, and the recombinant enzyme activity changed little when incubated in the buffer of pH8 - 10 on 37 degrees C for 1 h. The optimum reactive temprature was 75 degrees C, and the recombinant enzyme was more stable on the temperature of 25 - 55 degrees C. The half life of recombinant enzyme on 65 degrees C was 3.5 h, on 75 degrees C was 2.5 h. KmKG was 7.559 mmol/L, VmaxKG was 0.086 mmol/(L x min), KmAsp was 2.031 mmol/L, VmaxAsp was 0.024 mmol/(L x min). Ca2+, Fe3+, Mn2+ inhibited enzyme activity softly.
Aspartate Aminotransferases ; genetics ; isolation & purification ; metabolism ; Bacterial Proteins ; genetics ; isolation & purification ; metabolism ; Biocatalysis ; drug effects ; Electrophoresis, Polyacrylamide Gel ; Enzyme Stability ; drug effects ; Escherichia coli ; genetics ; Gene Expression Regulation, Bacterial ; Gene Expression Regulation, Enzymologic ; Hydrogen-Ion Concentration ; Kinetics ; Metals ; pharmacology ; Recombinant Proteins ; isolation & purification ; metabolism ; Temperature ; Thermus thermophilus ; enzymology ; genetics

Thermophilic bacteria strain YBJ-1 was isolated from hot spring samples collected from Yangbajing, Tibet. The 16sr DNA sequence of YBJ-1 (1511bp in length) shares 98% identity with that of Thermus scotoductus strain ITI-252T. The full-length ORF of amylase gene of YBJ-1 (amyT) was amplified by PCR technique and cloned into T-vector. The complete sequence of amyT is 1767bp in length, coding for 588 amino acids. The deduced amino acids share 99% similarity with alpha-cyclodextrinse of Bacillus sterothermophilus, 96% with maltogenic amylse of Thermus. sp IM6501, and 81% with neopullulanase of Bacillus sterothermophlus.
Amylases ; genetics ; Bacterial Proteins ; genetics ; Cloning, Molecular ; Culture Media ; Open Reading Frames ; genetics ; Thermus ; enzymology ; genetics ; isolation & purification

Genome of an extreme thermophile, Thermus caldophilus GK24 has been analyzed to construct the genomic map. The genomic DNAs encapsulated in agarose gel were digested with SspI, EcoRI, SpeI, and HpaI restriction endonucleases, and then the resulting genomic DNA fragments were analyzed by pulsed-field gel electrophoresis. Its restriction map has been constructed by analyzing sizes of the restriction fragments obtained from both complete and partial digestions. The circular form of its genome was composed of about 1.98 Mbp and a megaplasmid. The genomic loci for the genes of xylose isomerase, thioredoxin, tRNA-16S rRNA, 23S rRNA, L5 ribosomal protein, ADP-glucose pyrophosphorylase, DNA-ligase, and Tca DNA polymerase were determined by both Southern hybridization and PCR.
Chromosome Mapping* ; DNA ; DNA Restriction Enzymes ; Electrophoresis, Gel, Pulsed-Field ; Genome* ; Glucose-1-Phosphate Adenylyltransferase ; Polymerase Chain Reaction ; Ribosomal Proteins ; Sepharose ; Thermus* ; Thioredoxins ; Xylose

To express recombinant carboxypeptidase from Thermus aquaticus (Cpase Taq) in Pichia pastosis, the open reading frame coding thermostable Cpase Taq was optimized based on the preference of P. pastoris codon usage and synthesized in vitro. The novel gene was cloned into P. pastoris expression vector pHBM905A and the sequence coding 6xHis tag was fused with the ORF of Cpase Taq gene. The recombinant plasmid was named pHBM905A-Cpase Taq and transformed into P. pastoris GS 115. Transformants were induced with 1% methanol for 72 h until the enzyme yield reached 0.1 mg/ml. The enzyme was purified and its enzymatic properties were analyzed. The results showed that the specific enzyme activity reached maximum at 75 °C and pH 7.5, which was about 80 U/mg. It was the first report about the secretory expression of Cpase Taq in P. pastoris GS115. Because of its large-scale preparation, this enzyme may be applied in industrial hydrolysis of peptides into amino acids in the future.
Bacterial Proteins ; biosynthesis ; genetics ; Carboxypeptidases ; biosynthesis ; genetics ; Cloning, Molecular ; Codon ; Hydrolysis ; Open Reading Frames ; Pichia ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Thermus ; enzymology

Beta-glycosidase (Tngly) from the thermophilic eubacterium Thermus nonproteolyticus HG102, which is a thermostable monomeric protein and adopts the (beta/alpha)8 barrel fold, is an excellent model system to be investigated for the thermostable mechanism, activity and substrate specificity. Here, based on the analysis of structural basis for thermostability of Tngly (Wang et al, 2003) and comparison of other proteins structure of homofamily, Glu164 and Glu338 may act as proton donor and nucleophile in the hydrolysis reaction respectively; proline located at N1 of alpha-helix and arginine which can form ion link may contribute to the thermostability. We aim to further identify the critical sites and the amino acid residue(s) responsible for the activity, the thermal stability and the substrate specificity. Mutations had been constructed by site-directed mutagenesis. They are Glu164Gln, Glu338Ala, Pro316Gly, Arg325Leu, Pro344Phe, Pro356Ala and Pro316Gly/Pro356Ala. All mutant proteins were purified to SDS-PAGE purity. Changes in the conformations were examined by means of CD. The Glu338Ala mutant showed no detectable hydrolysis activity, but can synthesize oligosaccharides, as expected for the residue acting as the nucleophile of the reaction. The Glu164 acts as the general acid/base catalyst in the hydrolysis reaction. Changes in stabilities of mutants compared with wild-type were determined by means of heat inactivity experiment. These results indicate that the amino acid residue of proline that is located at N1 positions of alpha-helix, and Arg325 that form salt bridge between alpha-helices 5 and alpha-helices 6, are the critical sites to protein thermostabilization.
Bacterial Proteins ; genetics ; metabolism ; Enzyme Stability ; Hot Temperature ; Hydrolysis ; Mutagenesis, Site-Directed ; Mutation ; Structure-Activity Relationship ; Thermus ; enzymology ; genetics ; beta-Glucosidase ; genetics ; metabolism

By constructing the genomic DNA library of Meiothermus ruber CBS-01, the genes of trehalose phosphate synthase (TPS) and trehalose phosphate phosphatase (TPP) involved in trehalose synthesis were cloned. The genes were cloned into the plasmid pET21a, and expressed in Escherichia coli Rosetta gami (DE3). The activities of these two purified enzymes were confirmed by thin layer chromatography (TLC). Meanwhile, we tested the cellular compatible solutes of M. ruber CBS-01 under different environmental pressure, and found that under hyperosmotic pressure, this strain can accumulate trhalose-6-phosphate, but not trehalose. These results can give more insight to future research in the roles of TPS/TPP and TreS pathway.
Bacterial Proteins ; genetics ; metabolism ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Glucosyltransferases ; genetics ; metabolism ; Phosphoric Monoester Hydrolases ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; metabolism ; Thermus ; enzymology ; genetics ; Trehalose ; biosynthesis

The gene coding for beta-glycosidase (EC3.2.1.21) from Thermus nonproteolyticus HG102 has been cloned and expressed in E. coli. The gene open reading frame was 1311 bp and it codes for 436 amino acids. The deduced amino acid sequence of the enzyme showed identity with members of glycosyl hydrolase family I. The enzyme had high content of hydrophobic amino acid (Ala 12.8%, Leu 10.9%), Arg(9.6%), Glu(9.4%) and Pro(8.0%), but low content Cys(0.45%) and Met (0.9%). From the alignment of enzyme amino acid sequence with other glycosyl hydrolase family I members, Glu164 and Glu338 were predicated as the proton donor and nucleophile group. The DNASTAR program was used to predict the secondary structure. According to the Chou-Fasman model, the enzyme has 41.4% of alpha-helics, 16.2%, beta-strands, 14.4%, beta-turns. 14 of the 35 Pro were located at the second sites of beta-turns. Hydrophobic interaction, ion bond, alpha-helics and Pro had important contribution to Tn-gly thermostability.
Amino Acid Sequence ; Cloning, Molecular ; Escherichia coli ; genetics ; Glycoside Hydrolases ; biosynthesis ; classification ; genetics ; Hot Temperature ; Molecular Sequence Data ; Open Reading Frames ; genetics ; Phylogeny ; Protein Structure, Secondary ; physiology ; Recombinant Proteins ; biosynthesis ; genetics ; Sequence Analysis, DNA ; methods ; Sequence Homology ; Thermus ; enzymology ; beta-Glucosidase