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Several native and engineered heat-stable DNA polymerases from a variety of sources are used as powerful tools in different molecular techniques, including polymerase chain reaction, medical diagnostics, DNA sequencing, biological diversity assessments, and in vitro mutagenesis. The DNA polymerase from the extreme thermophile, Thermus scotoductus strain K1, (TsK1) was expressed in Escherichia coli, purified, and characterized. This enzyme belongs to a distinct phylogenetic clade, different from the commonly used DNA polymerase I enzymes, including those from Thermus aquaticus and Thermus thermophilus. The enzyme demonstrated an optimal temperature and pH value of 72-74°C and 9.0, respectively, and could efficiently amplify 2.5 kb DNA products. TsK1 DNA polymerase did not require additional K + ions but it did need Mg 2+ at 3-5 mM for optimal activity. It was stable for at least 1 h at 80°C, and its half-life at 88 and 95°C was 30 and 15 min, respectively. Analysis of the mutation frequency in the amplified products demonstrated that the base insertion fidelity for this enzyme was significantly better than that of Taq DNA polymerase. These results suggest that TsK1 DNA polymerase could be useful in various molecular applications, including high-temperature DNA polymerization., (© 2021 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Structural studies of -10 promoter element recognition by domain 2 of the RNA polymerase σ subunit [Feklistov & Darst (2011), Cell, 147, 1257-1269] reveal an unusual crystal-packing arrangement dominated by G-quartets. The 3'-terminal GGG motif of the oligonucleotide used in crystallization participates in G-quadruplex formation with GGG motifs from symmetry-related complexes. Stacking between neighboring G-quadruplexes results in the formation of pseudo-continuous four-stranded columns running throughout the length of the crystal (G-columns). Here, a new crystal form is presented with a different arrangement of G-columns and it is proposed that the fortuitous finding of G-quartet packing could be useful in engineering crystal contacts in protein-ssDNA complexes.

Succinyl-CoA synthetase (SCS) from Thermus aquaticus was characterized biochemically via measurements of the activity of the enzyme and determination of its quaternary structure as well as its stability and refolding properties. The enzyme is most active between pH 8.0 and 8.4 and its activity increases with temperature to about 339 K. Gel-filtration chromatography and sedimentation equilibrium under native conditions demonstrated that the enzyme is a heterotetramer of two α-subunits and two β-subunits. The activity assays showed that the enzyme uses either ADP/ATP or GDP/GTP, but prefers GDP/GTP. This contrasts with Escherichia coli SCS, which uses GDP/GTP but prefers ADP/ATP. To understand the nucleotide preference, T. aquaticus SCS was crystallized in the presence of GDP, leading to the determination of the structure in complex with GDP-Mn(2+). A water molecule and Pro20β in T. aquaticus take the place of Gln20β in pig GTP-specific SCS, interacting well with the guanine base and other residues of the nucleotide-binding site. This leads to the preference for GDP/GTP, but does not hinder the binding of ADP/ATP.

Succinyl-CoA synthetase (SCS) is an enzyme of the citric acid cycle and is thus found in most species. To date, there are no structures available of SCS from a thermophilic organism. To investigate how the enzyme adapts to higher temperatures, SCS from Thermus aquaticus was cloned, overexpressed, purified and crystallized. Attempts to crystallize the enzyme were thwarted by proteolysis of the beta-subunit and preferential crystallization of the truncated form. Crystals of full-length SCS were grown after the purification protocol was modified to include frequent additions of protease inhibitors. The resulting crystals, which diffract to 2.35 A resolution, are of the protein in complex with Mn2+-GDP.

A DNA aptamer specific for Thermus aquaticus DNA polymerase (Taq-polymerase) was immobilized on magnetic beads, which were prepared in the presented study. The effect of various parameters including pH, temperaturem and aptamer concentration on the immobilization of 5'-thiol labeled DNA-aptamer onto glutaric dialdhyde activated magnetic beads was evaluated. The binding conditions of Taq-polymerase on the aptamer immobilized magnetic beads were studied using commercial Taq-polymerase to characterize the surface complexation reaction. Efficiency of affinity magnetic beads in the purification of recombinant Taq-polymerase from crude extracts was also evaluated. For this case, the enzyme "recombinant Taq-DNA polymerase" was cloned and expressed using an Amersham E. coli GST-Gene Fusion Expression system. Crude extracts were in contact with affinity magnetic beads for 30 min and were collected by magnetic field application. The purity of the eluted Tag-polymerase from the affinity beads, as determined by HPLC, was 93% with a recovery of 89% in a one-step purification protocol. Apparently, the system was found highly effective as one step for the low-cost purification of Taq-polymerase in bacterial crude extract.

A quantitative screening for intra- and extracellular lipolytic activity was performed in submerged cultures of four Thermus strains using two different media (named T or D medium). Major differences in the extracellular lipolytic activity were observed in T medium, the highest values being for Thermus thermophilus HB27 and Thermus aquaticus YT1 strains (18 and 33 U/L, respectively). Two enzymes with lipase/esterase activity were identified in the four Thermus strains by zymogram analysis, with molecular weights of 34 and 62 kDa. No kinetic typification of the enzymes as primary metabolites was possible for any of the Thermus strains, because of the lack of a good fitting of the experimental lipolytic activity production rates to the Luedecking and Piret model. However, a linear relationship was found between the absolute values of biomass and total lipase/esterase activity (sum of intracellular and extracellular). For T. thermophilus HB27, an increase in the aeration rate caused the increase in the production of biomass and, particularly, intracellular lipolytic activity but the extracellular lipolytic activity was not affected except for the series with the strongest oxygen limitation. Transmission electronic microscopy revealed that T. thermophilus HB27 formed rotund bodies surrounded by a common membrane in cultures in the early stationary phase. The results suggest the occurrence of a specific mechanism of lipase/esterase secretion that might be due to the different composition and permeability of the cell membranes and those surrounding the rotund bodies.

The heating of protein preparations of mesophilic organism (e.g., E. coli) produces the obliteration of all soluble multimeric proteins from this organism. In this way, if a multimeric enzyme from a thermophilic microorganism is expressed in these mesophilic hosts, the only large protein remaining soluble in the preparation after heating is the thermophilic enzyme. These large proteins may be then selectively adsorbed on lowly activated anionic exchangers, enabling their full purification in just these two simple steps. This strategy has been applied to the purification of an alpha-galactosidase and a beta-galactosidase from Thermus sp. strain T2, both expressed in E. coli, achieving the almost full purification of both enzymes in only these two simple steps. This very simple strategy seems to be of general applicability to the purification of any thermophilic multimeric enzyme expressed in a mesophilic host.

Escherichia coli BL21 (DE3) transformed with a thermostable Thermus maltogenic amylase (ThMA), isolated from a Gram-negative bacterium Thermus strain IM6501, grew well and efficiently produced ThMA in a complex medium but not in a chemically defined medium (DM). By supplementing L-glutamate to DM medium, both the specific growth rate and ThMA expression significantly increased. Alterations in the cellular responses of recombinant E. coli to L-glutamate were analyzed at the protein level by two-dimensional gel electrophoresis and mass spectrometry. The ppGpp synthase (RelA) was significantly reduced in cells grown with L-glutamate and was consistent with the low level of ppGpp, an indicator of stringent response. On the other hand, protein chain elongation factor (EF-Tu) and manganese-containing superoxide dismutase (MnSOD), which protects cells against oxidative damage, was significantly elevated by L-glutamate supplementation. These results indicate that L-glutamate enhances ThMA expression and increases the E. coli growth rate not only by overcoming the stringent response but also by increasing the synthesis of EF-Tu and MnSOD.

This work exemplifies the advantages of using a battery of new heterofunctional epoxy supports to immobilize enzymes. We have compared the performance of a standard Sepabeads-epoxy support with other Sepabeads-epoxy supports partially modified with boronate, iminodiacetic, metal chelates, and ethylenediamine in the immobilization of the thermostable beta-galactosidase from Thermus sp. strain T2 as a model system. Immobilization yields depended on the support, ranging from 95% using Sepabeads-epoxy-chelate, Sepabeads-epoxy-amino, or Sepabeads-epoxy-boronic to 5% using Sepabeads-epoxy-IDA. Moreover, immobilization rates were also very different when using different supports. Remarkably, the immobilized beta-galactosidase derivatives showed very improved but different stabilities after favoring multipoint covalent attachment by long-term alkaline incubation, the enzyme immobilized on Sepabeads-epoxy-boronic being the most stable. This derivative had some subunits of the enzyme not covalently attached to the support (detected by SDS-PAGE). This is a problem if the biocatalysts were to be used in food technology. The optimization of the cross-linking with aldehyde-dextran permitted the full stabilization of the quaternary structure of the enzyme. The optimal derivative was very active in lactose hydrolysis even at 70 degrees C (over 1000 IU/g), maintaining its activity after long incubation times under these conditions and with no risk of product contamination with enzyme subunits.

The gene (bglT) encoding Tca beta-glycosidase (Thermus caldophilus GK24 beta-glycosidase) was overexpressed under the control of the trp promoter on a high-copy-number plasmid, pTRPES, in Escherichia coli W3110. The purified Tca beta-glycosidase enzyme was used in a galactosyl-transfer reaction to synthesize galacto-oligosaccharides from lactose. The optimum temperature and pH for the enzyme to synthesize galacto-oligosaccharides from 30% (w/v) lactose were 80 degrees C and 6.0, respectively. The major product of the reaction was a trisaccharide. The thermostable Tca beta-glycosidase produced galacto-oligosaccharides efficiently during the hydrolysis of lactose.

The GTPases Ffh and FtsY are components of the prokaryotic signal recognition particle protein-targeting pathway. The two proteins interact in a GTP-dependent manner, forming a complex that can be stabilized by use of the non-hydrolyzable GTP analog GMPPCP. Crystals of the complex of the NG GTPase domains of the two proteins have been obtained from ammonium sulfate solutions. Crystals grow with several different morphologies, predominately as poorly diffracting plates and needle clusters, but occasionally as well diffracting rods. It has been demonstrated that all forms of the crystals observed contain an intact complex. Diffraction data to 2.0 A resolution have been measured.

A novel thermo-alkali-stable catalase from Thermus brockianus was purified and characterized. The protein was purified from a T. brockianus cell extract in a three-step procedure that resulted in 65-fold purification to a specific activity of 5300 U/mg. The enzyme consisted of four identical subunits of 42.5 kDa as determined by SDS-PAGE and a total molecular mass measured by gel filtration of 178 kDa. The catalase was active over a temperature range from 30 to 94 degrees C and a pH range from 6 to 10, with optimum activity occurring at 90 degrees C and pH 8. At pH 8, the enzyme was extremely stable at elevated temperatures with half-lives of 330 h at 80 degrees C and 3 h at 90 degrees C. The enzyme also demonstrated excellent stability at 70 degrees C and alkaline pH with measured half-lives of 510 h and 360 h at pHs of 9 and 10, respectively. The enzyme had an unusual pyridine hemochrome spectrum and appears to utilize eight molecules of heme c per tetramer rather than protoheme IX present in the majority of catalases studied to date. The absorption spectrum suggested that the heme iron of the catalase was in a 6-coordinate low spin state rather than the typical 5-coordinate high spin state. A K(m) of 35.5 mM and a V(max) of 20.3 mM/min.mg protein for hydrogen peroxide was measured, and the enzyme was not inhibited by hydrogen peroxide at concentrations up to 450 mM. The enzyme was strongly inhibited by cyanide and the traditional catalase inhibitor 3-amino-1,2,4-triazole. The enzyme also showed no peroxidase activity to peroxidase substrates o-dianisidine and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), a trait of typical monofunctional catalases. However, unlike traditional monofunctional catalases, the T. brockianus catalase was easily reduced by dithionite, a characteristic of catalase-peroxidases. The above properties indicate that this catalase has potential for applications in industrial bleaching processes to remove residual hydrogen peroxide from process streams.

A thermostable beta-glycosidase (Tn-gly) from Thermus nonproteolyticus HG102 has been cloned and overexpressed in Escherichia coli. The recombinant enzyme, with a molecular mass of 48.9 kDa, was purified to homogeneity. It can hydrolyze a wide range of oligosaccharides and perform transglycosylation. Crystals of the recombinant enzyme were grown by the hanging-drop vapour-diffusion technique with MPD and NaCl as precipitants. They belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 66.7, b = 94.6, c = 176.5 A.

Glycerol kinase (GlpK) is an important enzyme which catalyzes the rate-limiting step in a central biochemical pathway involving glycerol metabolism. GlpK from the thermophile Thermus aquaticus has been overexpressed in glpK-deficient Escherichia coli and crystallized by the hanging-drop method. The crystal belongs to the cubic space group I23, with unit-cell parameters a = b = c = 163.94 (3) A. Native data were collected to 2.87 A resolution on a Cu Kalpha rotating-anode X-ray source.

Thermostable alkaline phosphatase from Thermus sp. 3041 has been expressed in Escherichia coli, purified and crystallized. The crystals belong to space group P2(1)22(1), with unit-cell parameters a = 57.7, b = 69.9, c = 111.5 A. Diffraction data were collected to 2.54 A with a completeness of 91.1% (87.8% for the last shell), an R(merge) value of 0.105 (0.312) and an I/sigma(I) value of 9.5 (3.6).

Fructose-1,6-bisphosphate aldolase (E.C. 4.1.2) catalyses the reversible cleavage of fructose-1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde-3-phosphate in the glycolytic pathway of prokaryote and eukaryote organisms. The enzyme was obtained from the extreme thermophile Thermus aquaticus and, in contrast to mesophilic aldolases, expresses maximal activity in the presence of Co(2+) as cofactor instead of Zn(2+). The purified recombinant protein was monodisperse according to dynamic light-scattering measurements. Crystals of recombinant native class II fructose-1,6-bisphosphate aldolase from T. aquaticus were obtained from two different starting conditions at low protein concentrations. Condition I, using the sitting-drop vapour-diffusion method, yielded monoclinic crystals having space group P2 and unit-cell parameters a = 99.5, b = 57.5, c = 138.6 A, beta = 90.25 degrees. Diffraction data were collected to 2 A resolution at beamline X8-C of the NSLS synchrotron-radiation source. Native and selenomethionine-substituted protein crystals were obtained from condition II by hanging-drop vapor diffusion. The tetragonal crystals of the native protein belong to the space group P4(1), with unit-cell parameters a = b = 88.8, c = 163.1 A, while those of the SeMet protein have space group I4(1), with unit-cell parameters a = b = 88.6, c = 164.1 A. A data set suitable for MAD phasing was collected to 2.6 A resolution at beamline X8-C of the NSLS synchrotron source.

A D-hydantoinase from Thermus sp. was expressed in Escherichia coli, purified to homogeneity and crystallized both as native and Se-Met labelled protein. The crystals belong to the orthorhombic space group C222(1), with unit-cell parameters a = 125.9, b = 215.8, c = 207.5 A. A three-wavelength MAD data set was collected to 2.5 A resolution and a native data set was collected to 1.7 A resolution. Crystal packing and self-rotation calculations led to the assumption of six protomers per asymmetric unit, corresponding to a V(M) value of 2.28 A(3) Da(-1) and a solvent content of 46%. As each protomer contains nine Se-Met residues, 54 selenium sites per asymmetric unit were present and could be unambigously located in the course of the MAD experiment. This selenium substructure is one of the largest selenium substructures that have been solved to date. The resulting phases obtained at a high-resolution limit of 3.0 A could be extended to 1.7 A and refined by application of density-modification techniques, especially non-crystallographic symmetry.

A highly thermostable DNA ligase from Thermus filiformis has been crystallized at room temperature using methoxypolyethylene glycol 5000 as a precipitant. The crystal belongs to the monoclinic space group P2(1), with unit-cell parameters a = 90.63, b = 117.80, c = 98. 65 A, beta = 115.56 degrees. Two molecules of DNA ligase are present in the asymmetric unit, giving a crystal volume per protein mass (V(m)) of 3.1 A(3) Da(-1) and a solvent content of 61%. A native data set extending to 3.0 A resolution has been collected at 100 K using synchrotron X-rays.

Thermus aquaticus DNA polymerase I consists of the polymerase, the structure-specific nuclease and the vestigial editing nuclease domains. Three-dimensional structures of the native enzyme and its complex with DNA have already been reported. The structure of a complex with an inhibitory antibody has also been determined. The structure of the native enzyme in a different crystal form determined at 2.6 A is reported here. Optimized anomalous diffraction measurements made at the holmium L(III) edge were valuable in validating solutions obtained through molecular replacement. The structure of the polymerase domain is similar to those reported previously, while the relative orientation of the structure-specific nuclease domain is significantly different from those of the native enzyme and the DNA complex; it is, however, identical to that observed in the structure of the Fab complex. In the structures of the native enzyme and of the DNA complex reported previously, the active site of the structure-specific nuclease domain is too far from that of the polymerase domain, making it difficult to propose a structural model for the in vivo primer-excision and nick-translation activities of the enzyme. In the present structure, the two active sites are considerably closer. Taken together, the reported structure of the native enzyme, that of the Fab complex and the present structure imply that the different orientation of the structure-specific nuclease domain is probably a consequence of intrinsically high relative mobility between these two domains in this enzyme.

The gene encoding Thermus filiformis (Tfi) DNA polymerase was expressed under the control of the tac promoter on a high-copy plasmid, pJR, in Escherichia coli. The Tfi DNA polymerase was purified by using heat treatment and DEAE-Sephacel column chromatography. The purified enzyme had a molecular mass of 92 kDa, as estimated by SDS/PAGE. The optimum pH and temperature of the enzyme were 8.4-9.0 and 70-72.5 degrees C respectively. The half-life of the enzyme at 94 degrees C was approx. 40 min. The enzyme was activated by the bivalent cations, Mg(2+) and Mn(2+), and was inhibited by EDTA. The optimal Mg(2+) concentration of the enzyme was 4 mM. The optimal conditions for the PCR reaction were slightly different from those for the enzyme activity except for the optimal Mg(2+) concentration. Low concentrations of KCl had no effect on either the enzymic activity or the PCR amplification. The result of the PCR experiment with the enzyme indicates that Tfi DNA polymerase might be useful in DNA amplification.

An extremely thermostable UDP-GlcNAc pyrophosphorylase has been purified from Thermus caldophilus GK24 by chromatographic methods including ion-exchange, hydrophobic interaction, and affinity chromatographies. The specific activity of the enzyme was enriched 41.8-fold, with a recovery of 2%. The molecular mass of the enzyme was 41 kDa by SDS/PAGE and 45 kDa by gel-filtration chromatography. The activity was maximum at 86 degrees C and its half-life at 95 degrees C was 30 min. Its optimum pH was 6.9 in the presence of Mg2+ ions. A biochemical study showed that UDP-GlcNAc pyrophosphorylase activity could be enhanced by fructose 1-phosphate, a precursor of UDP-GlcNAc. The enzyme showed a broad substrate specificity with sugar 1-phosphates, including glucose 1-phosphate, GlcNAc-1-P and xylose 1-phosphate. The enzyme was therefore named UDP-sugar pyrophosphorylase. The N-terminal and internal peptide sequences were determined and compared with known sequences from various sources. It was found that N-terminal sequence is similar to that of UDP-GlcNAc and UDP-glucose pyrophosphorylases from other bacterial sources.

A highly thermostable xylose isomerase from Thermus caldophilus has been expressed in Escherichia coli. The purified enzyme has an optimum temperature of 363 K. It has been crystallized at room temperature using ammonium sulfate as a precipitant. The crystal belongs to the orthorhombic space group P212121, with unit-cell parameters a = 84.35, b = 123.60, c = 140.24 A. The presence of one molecule of tetrameric xylose isomerase in the asymmetric unit gives a crystal volume per protein mass (Vm) of 2.1 A3 Da-1 and a solvent content of 41% by volume. The crystals initially showed diffraction to 1.7 A Bragg spacing with synchrotron X-rays, and a set of native data extending to 2.3 A resolution has been collected.

NADH oxidase from Thermus aquaticus is a thermostable flavoenzyme that is similar in amino-acid sequence and other properties to the flavoenzyme component of the NADH peroxidase systems from Salmonella typhimurium and Amphibacillus xylanus. The enzyme has been isolated from T. aquaticus and crystallized using the hanging-drop method of vapour diffusion with sodium citrate as a precipitant at pH 8.5. The crystals belong to the hexagonal space group P622 with unit-cell dimensions a = b = 89.9, c = 491.6 A, and diffract to 2.5 A resolution.