A soluble NADH dehydrogenase (NADH: ferricyanide oxidoreductase) from Thermus aquaticus strain T351.

A soluble NADH dehydrogenase (NADH:ferricyanide oxidoreductase) has been obtained by simple disruption of cells of Thermus aquaticus strain T351, and purified. The enzyme is of low molecular mass, 50 000 Da, and displays many of the properties of the membrane-bound enzyme, including inhibition by both NADH and ferricyanide, and the same Km for ferricyanide. The enzyme contains 0.05 mol of FMN, 0.16 mol of labile sulphur and 2.2 mol of iron per mol of protein. The enzyme is inhibited by NAD and cupferron competitively with ferricyanide, and by ATP (but not ADP) competitively with NADH. The enzyme is particularly thermostable, having a half-life at 95 degrees C of 35 min. The effect of temperature on the molar absorption coefficient and the stability of NADH was determined.     

Purification and characterization of a thermostable proteinase isolated from Thermus sp. strain Rt41A.

Thermus sp. strain Rt41A produces an extracellular thermostable alkaline proteinase. The enzyme has a high isoelectric point (10.25-10.5) which can be exploited in purification by using cation-exchange chromatography. The proteinase was purified to homogeneity and has a molecular mass of 32.5 kDa by SDS/PAGE. It is a glycoprotein, containing 0.7% carbohydrate as glucose equivalents, and has four half-cystine residues present as two disulphide bonds. Maximum proteolytic activity was observed at pH 8.0 against azocasein and greater than 75% of this activity was retained in the pH range 7.0-10.0. Substrate inhibition was observed with casein and azocasein. The enzyme was stable in the pH range 5.0-10.0 and maximum activity, in a 10-min assay, was observed at 90 degrees C with 5 mM CaCl2 present. No loss of activity was observed after 24 h at 70 degrees C and the half-lives at 80 degrees C and 90 degrees C were 13.5 h and 20 min, respectively. Removal of Ca2+ reduced the temperature for maximum proteolytic activity against azocasein to 60 degrees C and the half-life at 70 degrees C was 2.85 min. The enzyme was stable at low and high ionic strength and in the presence of denaturing reagents and organic solvents. Rt41A proteinase cleaved a number of synthetic amino acid p-nitrophenol esters, the kinetic data indicating that small aliphatic or aromatic amino acids were the preferred residue at the P1 position. The kinetic data for the hydrolysis of a number of peptide p-nitroanilide substrates are also reported. Primary cleavage of the oxidized insulin B chain occurred at sites where the P1' amino acid was aromatic. Minor cleavage sites (24 h incubation) were for amino acids with aliphatic side chains at the P1' position. The esterase and insulin cleavage data indicate the specificity is similar for both the P1 and P1' sites.     

Scaling-up the production of thermostable lipolytic enzymes from Thermus aquaticus YT1

The lipolytic enzymes synthesized by Thermusaquaticus YT1 present extremely interesting properties of thermostability (more than 70% of activity after 12 days at 80°C and a half-life time of 1 h at 95°C), which point out the interest of proposing efficient strategies to successfully tackle the scale-up of the production process. In this study,viable scaling-up of the production process was implemented,and relevant aspects affecting the enzyme synthesis, such as the mineral composition of the culture medium, the aeration and the agitation have been evaluated.A strategy combining the modification of the culture medium and the aeration degree was also approached by adding perfluorocarbons, compounds which improve the availability of oxygen in the culture medium. An opposite response of biomass and lipolytic activity to the aeration conditions was found between scales (about 600 U L(-1) at high aeration levels in flask vs. 150 U L(-1) at high aeration rates in reactor), which further demonstrates the important role of the hydrodynamic conditions on the suitable development of the biological process. In all cases, the cultures were kinetically characterized and the Luedeking and Piret model turned out to be a valuable tool to conclude that the produced lipolytic enzyme is a growth-associated metabolite, no matter the medium and the scale.     

Purification and characterization of a thermostable carboxypeptidase (carboxypeptidase Taq) from Thermus aquaticus YT-1.

A thermostable carboxypeptidase, which we named carboxypeptidase Taq, was purified from Thermus aquaticus YT-1 and characterized. The molecular weight of the enzyme was estimated to be about 56,000 and 58,000 on SDS-polyacrylamide gel electrophoresis and gel filtration, respectively, indicating that the enzyme has a monomeric structure. The optimum pH of the enzyme was 8.0, and the optimum temperature for the reaction was 80 degrees C. The enzyme activity was dependent on cobalt ion and was inhibited by metal-chelating reagents, indicating that the enzyme is a metalloenzyme. The enzyme had high thermostability independent of cobalt ion; about 90% of its activity remained even after treatment at 80 degrees C for 5 h. The enzyme showed broad substrate specificity, although proline at the C-terminus of peptides was not cleaved. The enzyme released amino acids sequentially from the C-terminus.     

Novel thermostable ssDNA-binding proteins from Thermus thermophilus and T. aquaticus-expression and purification

Single-stranded DNA-binding proteins (SSBs) play essential roles in DNA replication, recombination, and repair in bacteria, archaea, and eukarya. We report here the identification, expression, and purification of the SSB-like proteins of the thermophilic bacteria Thermus thermophilus and T. aquaticus. The nucleotide (nt) sequence revealed that T. thermophilus SSB (TthSSB) and T. aquaticus (TaqSSB) consist of 264 and 266 amino acids, respectively, and have a molecular weight of 29.87 and 30.03kDa, respectively. The homology between these protein, is very high-82% identity and 90% similarity. They are the largest known prokaryotic SSB proteins. TthSSB and TaqSSB monomers have two putative ssDNA-binding sequences: N-terminal (located in the region from amino acids 1 to 123) and C-terminal (located between amino acids 124 and 264 or 266 in TthSSB and TaqSSB, respectively). PCR-derived DNA fragment containing the complete structural gene for TthSSB or TaqSSB protein was cloned into an expression vector. The clones expressing SSB-like proteins were selected and cloned DNA fragments were verified to be authentic by sequencing several clones. The purification was carried out using reduction of contamination by the host protein with heat treatment, followed by QAE-cellulose and ssDNA-cellulose column chromatography. We found our expression and purification system to be quite convenient and efficient, and will use it for production of thermostable SSB-proteins for crystallography study. We have applied the use of TthSSB and TaqSSB protein to increase the amplification efficiency with a number of diverse templates. The use of SSB protein may prove to be generally applicable in improving the PCR efficiency.     

Weakly bound calcium ions involved in the thermostability of aqualysin I, a heat-stable subtilisin-type protease of Thermus aquaticus YT-1.

Aqualysin I is a heat-stable protease; in the presence of 1 mM Ca(2+), the enzyme is stable at 80 degrees C and shows the highest activity at the same temperature. After gel filtration to remove free Ca(2+) from the purified enzyme sample, the enzyme (holo-aqualysin I) still bound Ca(2+) (1 mol/mol of the enzyme), but was no longer stable at 80 degrees C. On treatment of the holo-enzyme with EDTA, bound Ca(2+) decreased to about 0.3 mol/mol of the enzyme. The thermostability of holo-aqualysin I was dependent on the concentration of added Ca(2+), and 1 mM added Ca(2+) stabilized the enzyme completely, suggesting that aqualysin I has at least two Ca(2+) binding sites, i.e. stronger and weaker binding ones. Titration calorimetry showed single binding of Ca(2+) to the holo-enzyme with an association constant of 3.1 x 10(3) M(-1), and DeltaH and TDeltaS were calculated to be 2.3 and 6.9 kcal/mol, respectively, at 13 degrees C. La(3+), Sr(2+), Nd(3+), and Tb(3+) stabilized the holo-enzyme at 80 degrees C, as Ca(2+) did. These results suggest that the weaker binding site exhibits structural flexibility to bind several metal cations different in size and valency, and that the metal binding to the weaker binding site is essential for the thermostability of aqualysin I.     

Interactions of metal ions with mu-monothiopyrophosphate.

mu-Monothiopyrophosphate (MTP) binds monovalent and divalent metal ions with dissociation constants (Kd) similar to those for pyrophosphate (PPi). The values of Kd for metal-MTP complexes are the following, as measured kinetically in the hydrolysis of MTP (microM): Mg2+, 32 +/- 4; Mn2+, 5.4 +/- 1.4; and Co2+, 27 +/- 15. The thermodynamically measured (EPR) values for Mg2+ and Co2+ are 28 +/- 13 microns and 11 +/- 4 microM, respectively; and the Kd for the complex MnPPi is 3.4 +/- 0.5 microM. The metal-MTP complexes undergo hydrolysis at rates modestly faster or slower than the rate at which MTP itself reacts. The complexes MgMTP2-, CoMTP2-, and MnMTP2- undergo hydrolytic cleavage with release of thiophosphate with observed first-order rate constants of 1.6 x 10(-2) min-1, 2.3 x 10(-2) min-1, and 0.6 x 10(-2) min-1, respectively, at 35 degrees C, compared with 1.1 x 10(-2) min-1 for MTP4- under the same conditions. Alkali metal cations also stimulate or retard the hydrolysis of MTP. At 25 degrees C and pH 12.2, the observed rate constant for tetramethylammonium MTP4- is 2.1 x 10(-3) min-1, and the estimated rate constants (min-1) for saturating alkali metals under the same conditions are as follows: Li+, 0.25 x 10(-3); Na+, 3.9 x 10(-3), K+, 6.7 x 10(-3); and Cs+, 6.7 x 10(-3). Divalent metal ions markedly retard the hydrolysis of MTP at pH 7 and 8 because complexation shifts the pH rate profile more than 2 pH units toward the acid side.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus.

A stable deoxyribonucleic acid (DNA) polymerase (EC 2.7.7.7) with a temperature optimum of 80 degrees C has been purified from the extreme thermophile Thermus aquaticus. The enzyme is free from phosphomonoesterase, phosphodiesterase and single-stranded exonuclease activities. Maximal activity of the enzyme requires all four deoxyribonucleotides and activated calf thymus DNA. An absolute requirement for divalent cation cofactor was satisfied by Mg2+ or to a lesser extent by Mn2+. Monovalent cations at concentrations as high as 0.1 M did not show a significant inhibitory effect. The pH optimum was 8.0 in tris(hydroxymethyl)aminomethane-hydrochloride buffer. The molecular weight of the enzyme was estimated by sucrose gradient centrifugation and gel filtrations on Sephadex G-100 to be approximately 63,000 to 68,000. The elevated temperature requirement, small size, and lack of nuclease activity distinguish this polymerase from the DNA polymerase of Escherichia coli.     

Regulatory characteristics of phosphoenolpyruvate carboxylase from the extreme thermophile, Thermus aquaticus.

Phosphoenolpyruvate carboxylase from the extremely thermophilic bacterium, Thermus aquaticus YT-1, exhibits a virtually absolute requirement for acetyl CoA and there is strong positive cooperativity in the interaction of this activator with the enzyme. Several tricarboxylic acid cycle intermediates inhibit the enzyme. These findings suggest an anaplerotic role for the enzyme and an allosteric modulation of its activity by acetyl CoA and tricarboxylic acid cycle intermediates.     

Interactions of replication versus repair DNA substrates with the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus

Different DNA polymerases partition differently between replication and repair pathways. In this study we examine if two Pol I family polymerases from evolutionarily distant organisms also differ in their preferences for replication versus repair substrates. The DNA binding preferences of Klenow and Klentaq DNA polymerases, from Escherichia coli and Thermus aquaticus respectively, have been studied using a fluorescence competition binding assay. Klenow polymerase binds primed-template DNA (the replication substrate) with up to 50× higher affinity than it binds to nicked DNA, DNA with a 2 base single-stranded gap, blunt-ended DNA, or to a DNA end with a 3′ overhang. In contrast, Klentaq binds all of these DNAs almost identically, indicating that Klenow has a stronger ability to discriminate between replication and repair substrates than Klentaq. In contrast, both polymerases bind mismatched primed-template and blunt-ended DNA tighter than they bind matched primed-template DNA, suggesting that these two proteins may share a similar mechanism to identify mismatched DNA, despite the fact that Klentaq has no proofreading ability. In addition, the presence or absence of 5′- or 3′-phosphates has slightly different effects on DNA binding by the two polymerases, but again reinforce Klenow's more effective substrate discrimination capability.     

Electron image analysis of ribosomal subunits from Thermus aquaticus.

Electron micrographs of ribosomal subunits from the thermophilic bacterium Thermus aquaticus were analysed using multivariate statistical analysis and characteristic views constructed to reproducible spatial resolutions ranging from 1.9 to 3.6 nm. These views were comparable to morphological classes of Escherichia coli ribosomal subunits, albeit with differences in fine features also found in archaebacterial ribosomes.     

Cloning and expression of the thermostable pullulanase gene from Thermus sp. strain AMD-33 in Escherichia coli

Abstract The gene coding for a thermostable pullulanase from a thermophile, Thermus sp. strain AMD-33, was cloned in Escherichia coli using pDR540 as a vector. A restriction map was determined for the plasmid pTPS131 which contained the fragment carrying the pullulanase gene. DNA-DNA hybridisation analysis showed that the DNA fragment contained the gene from Thermus sp. strain AMD-33. The strain of E. coli harbouring the plasmid pTPS131 produced most of the pullulanase protein cellularly, whereas Thermus sp. strain AMD-33 produced pullulanase extracellularly. Comparative studies of the enzyme from the thermophile and the plasmid-encoded enzyme in E. coli demonstrated that the optimum temperature and pH of the enzymes were closely similar.     

Purification and characterization of a repressible alkaline phosphatase from Thermus aquaticus.

A repressible alkaline phosphatase has been isolated from the extreme bacterial thermophile, Thermus aquaticus. The enzyme can be derepressed more than 1,000-fold by starving the cells for phosphate. In derepressed cells, nearly 6% of the total protein in a cell-free enzyme preparation is alkaline phosphatase. The enzyme was purified to homogeneity as judged by disc acrylamide electrophoresis and sodium dodecyl sulfate electrophoresis. By sucrose gradient centrifugation it was established that the enzyme has an approximate molecular weight of 143,000 and consists of three subunits, each with a molecular weight of 51,000. Tris buffer stimulates the activity of the enzyme, which has a pH optimum of 9.2. The enzyme has a broad temperature range with an optimum of 75-80 degrees. The enzyme catalyzes the hydrolysis of a wide variety of phosphorylated compounds as do many of the mesophilic alkaline phosphatases. The Michaelis constant(Km) for the enzyme is 8.0 X 10(-4) M. Amino acid analysis of the protein revealed little in the amino acid composition to separate it from other mesophilic enzymes which have been previously studied.     

The role of bound calcium ions in thermostable, proteolytic enzymes. II. Studies on thermolysin, the thermostable protease from Bacillus thermoproteolyticus.

The functional properties of the four calcium ions, bound by thermolysin, appear to be very similar to those of the single calcium ion bound by thermomycolase (G. Voordouw and R.S. Roche (1975), Biochemistry, preceding paper in this issue). Hence when the free calcium ion concentration is varied in the range where the calcium double-site dissociates (G. Voordouw and R.S. Roche (1974), Biochemistry 13, 5017), no changes are observed in the sedimentation coefficient or the peptide circular dichroism. Differences in molar ellipticity and molar extinction coefficient occur in the aromatic ultraviolet region, which parallel the occupancy of the calcium binding double site. The difference spectrum, characterized by a main band at 290 nm and a somewhat smaller band at 283 nm, is interpreted as due to the transfer of a partially buried tryptophan residue to the aqueous solvent upon dissociation of the two calcium ions from the double site. This is most likely Trp-186, which is in between Asp-185 and Glu-187, two chelating amino acids of this site. From the calcium dependence of the rate constant for autolytic degradation we conclude, as for thermomycolase, that only conformers devoid of bound calcium ion serve as substrates in the reaction. This rate constant increases about 1000-fold, when the double site dissociates. Hydrogen-tritium exchange studies show the presence of a large stable strcutural core, comprising about 32% of all the peptide hydrogens present. These do not exchange-in after 24 hr at 25degreesC, pH 9.0, ionic strenth 0.1. The exchange-out of 60 slow hydrogens was found to be independent of the free calcium ion concentration in the range 2.0-8.0 X 10(-4) M, where all four calcium-binding sites are saturated. The calcium dependence of the first-order rate constant for thermal denaturation at 80degreesC, pH 7.0, indicates that thermolysin is stabilized by only one calcium ion under these conditions. These observations are rationalized in terms of a calcium-binding model for thermolysin and the known three-dimensional structure of the enzyme and its calcium-binding sites.     

Xylose (glucose) isomerase gene from the thermophile Thermus thermophilus: cloning, sequencing, and comparison with other thermostable xylose isomerases.

The xylose isomerase gene from the thermophile Thermus thermophilus was cloned by using a fragment of the Streptomyces griseofuscus gene as a probe. The complete nucleotide sequence of the gene was determined. T. thermophilus is the most thermophilic organism from which a xylose isomerase gene has been cloned and characterized. The gene codes for a polypeptide of 387 amino acids with a molecular weight of 44,000. The Thermus xylose isomerase is considerably more thermostable than other described xylose isomerases. Production of the enzyme in Escherichia coli, by using the tac promoter, increases the xylose isomerase yield 45-fold compared with production in T. thermophilus. Moreover, the enzyme from E. coli can be purified 20-fold by simply heating the cell extract at 85 degrees C for 10 min. The characteristics of the enzyme made in E. coli are the same as those of enzyme made in T. thermophilus. Comparison of the Thermus xylose isomerase amino acid sequence with xylose isomerase sequences from other organisms showed that amino acids involved in substrate binding and isomerization are well conserved. Analysis of amino acid substitutions that distinguish the Thermus xylose isomerase from other thermostable xylose isomerases suggests that the further increase in thermostability in T. thermophilus is due to substitution of amino acids which react during irreversible inactivation and results also from increased hydrophobicity.