Aquaticus YT-1 Research Articles

The third restriction-modification system from Thermus aquaticus YT-1: solving the riddle of two TaqII specificities.

Two restriction–modification systems have been previously discovered in Thermus aquaticus YT-1. TaqI is a 263-amino acid (aa) Type IIP restriction enzyme that recognizes and cleaves within the symmetric sequence 5′-TCGA-3′. TaqII, in contrast, is a 1105-aa Type IIC restriction-and-modification enzyme, one of a family of Thermus homologs. TaqII was originally reported to recognize two different asymmetric sequences: 5′-GACCGA-3′ and 5′-CACCCA-3′. We previously cloned the taqIIRM gene, purified the recombinant protein from Escherichia coli, and showed that TaqII recognizes the 5′-GACCGA-3′ sequence only. Here, we report the discovery, isolation, and characterization of TaqIII, the third R–M system from T. aquaticus YT-1. TaqIII is a 1101-aa Type IIC/IIL enzyme and recognizes the 5′-CACCCA-3′ sequence previously attributed to TaqII. The cleavage site is 11/9 nucleotides downstream of the A residue. The enzyme exhibits striking biochemical similarity to TaqII. The 93% identity between their aa sequences suggests that they have a common evolutionary origin. The genes are located on two separate plasmids, and are probably paralogs or pseudoparalogs. Putative positions and aa that specify DNA recognition were identified and recognition motifs for 6 uncharacterized Thermus-family enzymes were predicted.

TANGENTIAL FLOW MICROFILTRATION OF THERMUS AQUATICUS YT-1 LYSATE

The tangential flow microfiltration of Thermus aquaticus YT-1 lysate was investigated. Two membranes of different pore diameter – 0.2 μm PES and 0.65 μm PVDF – were compared. The optimal transmembrane pressure at different flow rates for both membranes was determined. Parameters such as flux through membrane, retention coefficients, rate constants and yields of nucleic acids, polysaccharides, proteins and product Taq DNA polymerase were evaluated. The flux of 0.2 μm pore diameter membrane was shown to be 1.35 times higher as compared with the 0.65 μm pore diameter membrane. A comparison of Taq DNA polymerase yield has revealed the majority of the product to flow through the membrane, but the highest yield of the product (96.3 ±3.7%) has been obtained with the 0.2 μm pore diameter membrane. The type of membrane material was shown to influence the performance of the process. It has been concluded that the 0.2 μm pore diameter membrane is suitable for the clarification of Thermus aquaticus YT-1 lysate. DOI: http://dx.doi.org/10.5755/j01.ct.59.1.1530

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.

A Novel Exopolysaccharide from the Biofilm of Thermus aquaticus YT-1 Induces the Immune Response through Toll-like Receptor 2

Bacterial polysaccharides are known to induce the immune response in macrophages. Here we isolated a novel extracellular polysaccharide from the biofilm of Thermus aquaticus YT-1 and evaluated its structure and immunomodulatory effects. The size of this polysaccharide, TA-1, was deduced by size-exclusion chromatography as 500 kDa. GC-MS, high performance anion-exchange chromatography with pulsed amperometric detection, electrospray ionization-MS/MS, and NMR revealed the novel structure of TA-1. The polysaccharide is composed of tetrasaccharide-repeating units of galactofuranose, galactopyranose, and N-acetylgalactosamine (1:1:2) and lacked acidic sugars. TA-1 stimulated macrophage cells to produce the cytokines TNF-α and IL-6. Screening of Toll-like receptors and antibody-blocking experiments indicated that the natural receptor of TA-1 in its immunoactivity is TLR2. Recognition of TA-1 by TLR2 was confirmed by TA-1 induction of IL-6 production in peritoneal macrophages from wild-type mice but not from TLR2(-/-) mice. TA-1, as a TLR2 agonist, could possibly be used as an adjuvant and could enhance cytokine release, which increases the immune response. Furthermore, TA-1 induced cytokine release is dependent on MyD88/TIRAP.

On the hyperthermostability of lipolytic enzymes from Thermus aquaticus YT‐1: exploring their application to polymer degradation

AbstractBACKGROUND: The investigation of enzymes from extremophilic microorganisms has generated great scientific interest in recent decades owing to their ability to operate in harsh conditions. Since lipolytic enzymes are one of the three groups most commercially demanded, the study of novel thermostable lipolytic enzymes is of much interest.RESULTS: Lipolytic enzymes produced by Thermus aquaticus YT1 have been investigated. Very interesting properties against thermal inactivation, with a half‐life time of 1 h at 95 °C were found. The use of compounds such as polyols is a strategy used to further improve the thermostability of these enzymes, and the modelling of the deactivation process with a series‐type equation, allows one to quantitatively ascertain the free energy of deactivation. Finally, the application to dibutyl adipate hydrolysis was investigated, as an indicator of the potential usefulness of the enzymes in polymer biodegradation.CONCLUSION: The crude lipolytic enzymes possess outstanding properties of thermostability (high lipolytic activity after 12 days at 80 °C, and a half‐life time of 1 h at 95 °C), and they can be significantly improved by adding polyols (addition of glycerol entailed activity losses of just 3% after 1 h at 95 °C). These enzymes are able to catalyze ester cleavage, showing a zero‐order kinetics. Copyright © 2011 Society of Chemical Industry

Production of Thermostable Lipolytic Activity by Thermus Species

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.

Stimulation of novel thermostable extracellular lipolytic enzyme in cultures of Thermus sp.

Selected organisms from the genus Thermus ( T. aquaticus YT1, T. thermophilus HB8 and HB27) offer new opportunities for biocatalysis and biotransformations as a result of the extreme stability of their enzymes. In order to favour the secretion of extracellular lipolytic enzymes the effect of temperature and carbon source has been studied. All strains were able to grow within a wide temperature range (from 60 to 80 °C) with an optimum value of 70 °C for extracellular lipolytic activity. On the other hand, several sugars were used as carbon source in the culture medium at different concentrations (from 0.5 to 14 g dm −3). Supplementation of monosaccharides (glucose, fructose) and disaccharides (maltose, sucrose) was carried out in an attempt to increase biomass and lipolytic enzyme production. The influence of carbon source was variable, depending on the strain. Nevertheless, the addition of low concentrations of mono- or disaccharides helped to improve the productivity of the process in most cases. The most significant effects on extracellular lipolytic activity were detected in T. thermophilus HB27 and T. aquaticus YT1 cultures, using sucrose and fructose as carbon source at an initial concentration of 0.5 g dm −3. Moreover, the effect of addition of lipids (olive oil, coconut oil and tributyrin) was tested. In T. thermophilus HB27 cultures, the highest extracellular lipolytic activities were obtained when olive oil was added (70% higher than those obtained in control cultures).

The action mode of Thermus aquaticus YT-1 4-α-glucanotransferase and its chimeric enzymes introduced with starch-binding domain on amylose and amylopectin

A thermostable 4-α-glucanotransferase (TAαGT) gene isolated from Thermus aquaticus YT-1 had an open reading frame of 1503 nucleotides, which encoded 500 amino acid residues for a 57,969 dalton protein. The maximum activity of the TAαGT was observed at pH 7.5 and 70 °C. The enzyme catalyzed intermolecular transglycosylation of maltooligosaccharides (disproportionation) to produce linear α-1,4-glucans of various sizes. The starch-binding domains (SBD) of Bacillus stearothermophilus ET1 CGTase (E and DE) were introduced into the C-terminus of TAαGT to enhance the starch utilizing activity. The chimeric enzymes, TAαGT-E and TAαGT-DE, showed no difference in temperature optimum, transglycosylation activity, and amylolytic degradation pattern compared to TAαGT wild-type. However, TAαGT-DE exhibited the highest molar specific activity toward amylose. TAαGT-DE modified amylopectin molecules by its disproportionating activities to produce modified amylopectin clusters ( M w 10 5–10 6). Also, it demonstrated the ability to produce cyclo-amyloses with DP of 19 through 35 from amylose molecules.

Role of the COOH-terminal pro-sequence of aqualysin I (a heat-stable serine protease) in its extracellular secretion by Thermus thermophilus.

Aqualysin I is a subtilisin-type serine protease secreted into the medium by Thermus aquaticus YT-1. Thermus thermophilus cells harboring a plasmid for the aqualysin I precursor secreted pro-aqualysin I with the C-terminal pro-sequence into the culture medium, and the precursor was then processed to the mature enzyme during the cultivation. However, the extracellular levels of aqualysin I in T. thermophilus cells harboring plasmids for deletion mutants as to the C-terminal pro-sequence were about 10-20% in comparison with the level of wild-type. Only the mature enzyme could be detected in the medium, while pro-aqualysin I with the C-terminal pro-sequence could not. These results suggest that the C-terminal pro-sequence of aqualysin I plays an important role in the extracellular secretion of aqualysin I.

1P408 Structure of Exopolysaccharide from the Biofilms of Thermus aquaticus YT-1(16. Cell-cell communication,Poster Session,Abstract,Meeting Program of EABS &BSJ 2006)

1P408 Structure of Exopolysaccharide from the Biofilms of Thermus aquaticus YT-1(16. Cell-cell communication,Poster Session,Abstract,Meeting Program of EABS &BSJ 2006)

Quantification of intra- and extra-cellular thermophilic lipase/esterase production by Thermus sp.

Four Thermus strains produced lipolytic activity when grown in liquid medium for 30 h at 70 degrees C. The highest total lipase/esterase activity (57 U l(-1)) was in Thermus aquaticus YT-1, followed by Thermus thermophilus HB27 and HB8 (33 and 25 U l(-1), respectively), and finally by Thermus sp. (16 U l(-1)). Extra-cellular activity was detected in T. aquaticus YT-1 and T. thermophilus HB27 (33 and 17 U l(-1)). All enzymes were stable at 80 degrees C over 30 min, and their activity towards fatty acid esters increased as substrate chain-length diminished (i.e. hydrolysis rate was up to 6-fold higher on p-nitrophenyl caproate than on laurate).

High-level expression, secretion, and purification of the thermostable aqualysin I from Thermus aquaticus YT-1 in Pichia pastoris

Aqualysin I is a heat-stable subtilisin-type serine protease which is secreted into the culture medium by Thermus aquaticus YT-1, an extreme thermophile. We report the high-level expression of an aqualysin I protein using its native signal sequence for secretion in the methylotrophic yeast, Pichia pastoris. The expression of aqualysin I in P. pastoris was carried out using the glyceraldehyde-3-phosphate dehydrogenase ( GAP) promoter. Pro-aqualysin I (38 kDa) as inactive protein was secreted into the medium of shake-flask cultures at a concentration of 1 g/L. It was isolated from the culture supernatant by an ammonium sulfate precipitation and one-step anion exchange chromatography in a nearly pure form and was autoproteolytically activiated by heat treatment. A proteolytic activity test indicated that the purified recombinant aqualysin I was properly folded with a specific activity similar to that of the native enzyme. We also explored the possibility of secreting the GAP expressed aqualysin I in P. pastoris by in-frame fusion of the Saccharomyces cerevisiae α-factor secretion signal. However, the levels of secreted pro-aqualysin I particles were approximately 10 times lower, possibly as a consequence of membrane association or to the influence of the α-factor secretion signal sequences on the transcription or secretion of aqualysin I. When considering further optimization of the downstream process and culture conditions for high-level production of recombinant aqualysin I by P. pastoris, this expression system is promising for development as an industrial process.

Independently expressed N-terminal pro-domain of aqualysin I precursor complements the folding of its mature domain to active form in Escherichia coli.

Aqualysin I is a subtilisin-type serine protease secreted into the culture medium by Thermus aquaticus YT-1. It is first produced as a large precursor that consists of a signal peptide, an N-terminal pro-domain, the mature protease domain and a C-terminal pro-domain. To investigate whether the N-terminal pro-domain supplied in trans as an independent peptide plays an important role in the folding and secretion of the protease, the N-terminal pro-domain in E. coli has been expressed independent of the mature domain with or without the C-terminal pro-domain using an expression system with separate promoters and signal peptides. Protease assay and SDS-PAGE clearly showed that the N-terminal pro-domain plays an essential role in guiding the proper folding in trans of the enzymatically active conformation of aqualysin I. The N-terminal amino acid sequences of the purified enzymes were identical and had no signal peptides. These results indicate that independently expressed domains are secreted into the periplasmic space before the N-terminal pro-domain-assisted folding of the mature domain.

Role of the C -terminal pro-domain of aqualysin I in the maturation and translocation of the precursor across the cytoplasmic membrane in Escherichia coli

Aqualysin I, which is a subtilisin-type, extracellular protease secreted by Thermus aquaticus YT-1, is synthesized as a unique precursor bearing pro-domains at both N- and C-terminus of the mature protease domain as well as an N-terminal signal peptide. To investigate the function of the C-terminal pro-domain in maturation and export pathway of the precursor in E. coli cells, aqualysin I variants were constructed in which deletion mutants of the C-terminal pro-domain lacking its own signal peptide were inserted into pIN-III-ompA3. When E. coli harboring wild type and mutant plasmids were induced by 0.2 mM IPTG, active aqualysin I was produced by heat treatment at 65 °C. Aqualysin I precursors with deletions of more than 5 amino acid residues at the C-terminal end of pro-domain were much more rapidly processed than that of wild type, indicating that the C-terminal pro-domain functions as a inhibitor for processing of aqualysin I precursor. With the wild type, most of aqualysin I was present in membrane fraction (probably the outer membrane), whereas for the truncated mutants, it remained in the cytoplasm, indicating that for deletion mutants, their precursors expressed in cells were not translocated across the cytoplasmic membrane, despite the existence of an N-terminal signal peptide.

Expression of a thermostable restriction endonuclease in recombinant Escherichia coli cells and optimization of fermentation conditions

Taq I restriction endonuclease gene of the thermophilic eubacterium Thermus aquaticus YT-1 (ATCC 25104) was successfully cloned and expressed in recombinant Escherichia coli cells under the control of the lac promoter/operator system. Higher Taq I endonuclease specific activities and biomass yields were obtained from E. coli ER2508(pUCTaq) cells when they were induced at the late-exponential phase of their growth. Taq I endonuclease expression was found to be host strain-dependent such that, among the three different strains examined, E. coli XL1(pUCTaq) produced the highest specific Taq I endonuclease activities for longer induction periods. Decreasing the inducer concentration from 1 to 0.1 mM not only improved the specific enzyme activity yields but also is more economical, considering the high cost of isopropyl-β-D-thiogalactopyranoside (IPTG). The optimum culture temperature was found to be 37 °C. Taq I endonuclease specific activity recovered from E. coli XL1(pUCTaq) cells was 935 U/mg under optimum conditions.

Molecular cloning, expression, purification, and characterization of fructose-1,6-bisphosphate aldolase from Thermus aquaticus.

Fructose-1,6-bisphosphate aldolase from the thermophilic eubacteria, Thermus aquaticus YT-1, was cloned and sequenced. Nucleotide-sequence analysis revealed an open reading frame coding for a 33-kDa protein of 305 amino acids having amino acid sequence typical of thermophilic adaptation. Multiple sequence alignment classifies the enzyme as a class II B aldolase that shares similarity with aldolases from other extremophiles: Thermotoga maritima, Aquifex aeolicus, and Helicobacter pylori (49--54% identity, 76--81% homology). Taq FBP aldolase was overexpressed under tac promoter control in Escherichia coli and purified to homogeneity using heat treatment followed by two chromatographic steps. Yields of 40--50 mg of monodisperse protein were obtained per liter of culture. The quaternary structure is that of a homotetramer stabilized by an apparent 21-amino-acid insertion sequence. The recombinant protein is thermostable for at least 45 min at 80 degrees C with little residual activity below 60 degrees C. Kinetic characterization at 70 degrees C, the optimal growth temperature for T. aquaticus, indicates extreme negative subunit cooperativity (h = 0.32) with a limiting K(m) of 305 microM. The maximal specific activity (V(max)) is 46 U/mg at 70 degrees C.

Identification and designing of the S3 site of aqualysin I, a thermophilic subtilisin-related serine protease.

Aqualysin I is a bacterial subtilisin-related alkaline serine protease, originating in Thermus aquaticus YT-1. Based on computational analysis, we predicted that two residues, Ser102 and Gly131, form the S3 site of aqualysin I, and we proved that this prediction by site-directed mutagenesis. To alter the P3-specificity of the enzyme, we built a "wall" on the S3 site edge by introducing a bulky side chain at target sites. Six mutant proteins were prepared: S102H, S102K, S102E, G131H, G131K, and G131D. The mutant enzymes were examined with two kinetically typical peptides for aqualysin I, suc-X-Ala-Ala-pNA, where X is Ala or Phe. All mutations reduced the efficiency for the Phe-containing peptide, while they raised the k(cat) values for the Ala-containing peptide. Especially, the S102K mutant protein hydrolyzed the polyalanine peptide efficiently. The strategies we have adopted in this paper are applicable to all subtilisin-related enzymes.

Identification, cloning and expression of p25, an AT-rich DNA-binding protein from the extreme thermophile, Thermus aquaticus YT-1.

Although the G+C content of Thermus aquaticus YT-1 chromosomal DNA is 67.4%, regions with lower G+C content have also been observed. AT-rich DNA-binding proteins may contribute to the thermostability and biological functions of these DNA regions at Thermus growth temperatures. Using double-stranded DNA (dsDNA)-cellulose chromatography, a T.aquaticus YT-1 protein, designated as p25, was identified to bind preferentially to AT-rich DNA. The gene encoding p25 was cloned and sequenced after immunoscreening T.aquaticus YT-1 expression libraries. The deduced primary structure of p25 is 211 amino acids in length with a molecular weight of 23 225 Da. Native p25 was purified and characterized as a homodimer with modification possibly at lysine and arginine residues. Its preferential and temperature-dependent binding to AT-rich DNA was confirmed with mobility-shift DNA-binding assays. The protein was demonstrated to bind preferentially to dsDNA instead of single-stranded DNA. The binding of p25 to dsDNA also improved the thermotolerence of this protein. Overexpression study of fusion p25 suggested that the N-terminus of the protein might form the DNA-binding domain or be closely involved in DNA-binding activity.

Substrate Specificity of Aqualysin I Altered by an Organic Solvent, DMSO.

Aqualysin I is the alkaline serine protease isolated from an extreme thermophile, Thermus aquaticus YT-1. We have analyzed the kinetic properties of aqualysin I, using thirty-one kinds of chromogenic succinyl-tripeptide p-nitroanilides as substrates in the presence of 10% dimethylsulfoxide (DMSO). Aqualysin I hydrolyzed many peptides in a DMSO-containing mixture, however the substrate specificity was different from that in the absence of DMSO. The Km for each peptide was raised by the addition of 10% DMSO. Also, the P3- as well as P2-specificity of aqualysin I was altered. These results suggested that the side chains of the P2 and P3 residues are exposed to the solvent, and the hydrophobic interactions between the side chain of the substrate and the solvent may take a part in the substrate recognition of the enzyme.

F- and V-ATPases in the Genus Thermus and Related Species

The discovery of a V-type ATPase in the gram-negative bacterium Thermus thermophilus HB8 (YOKOYAMA et al., J. Biol. Chem. 265, 21946, 1990) was unexpected, since only eukaryotic endomembranes and archaea were thought to contain this enzyme complex, and horizontal gene transfer was suggested to explain the finding. We examined membrane-associated ATPases from representatives of several groups of the genus Thermus. The enzymes were extracted with chloroform and purified by ion exchange chromatography or native gel electrophoresis. One novel Islandic isolate, T. scotoductus SE-1, as well as strain T. filiformis from New Zealand, possessed F-ATPases, as judged by the typical five subunit composition of the F1-moiety, sensitivity to azide, insensitivity to nitrate and a strong crossreaction with antibodies against the F1-ATPase from E. coli. In addition, N-terminal amino acid sequencing of the beta subunit from T. scotoductus SE-1 confirmed its homology with beta subunits from known F-ATPases. In contrast, the same extraction procedure released a V-ATPase from the membranes of T. thermophilus HB27 and T. aquaticus YT-1. The related species Meiothermus (formerly Thermus) chliarophilus ALT-8 also possessed a V-ATPase. All V-ATPases examined in this study contained larger major subunits than F-ATPases, crossreacted with antiserum against subunit A of the V-ATPase from the archaeon Halobacterium saccharovorum, and the N-terminal sequences of their major subunits were homologous to those of other V-ATPases. Sequences of the 16S rRNA gene clearly placed T. scotoductus SE-1, along with other non-pigmented Thermus strains, as a distinct species close to T. aquaticus. Our results suggested that at least two members of the genus, T. scotoductus SE-1 and T. filiformis, contain an F-ATPase, whereas several others possess a V-ATPase. These data could indicate a greater diversity of the genus Thermus than was previously thought. Alternatively, the genus may consist of species where horizontal gene transfer has occurred and others, where it has not.