Hyperthermophilic Bacterium Thermotoga Neapolitana Research Articles

Occurrence of Capnophilic Lactic Fermentation in the Hyperthermophilic Anaerobic Bacterium Thermotoga sp. Strain RQ7

Capnophilic lactic fermentation (CLF) is an anaplerotic pathway exclusively identified in the anaerobic hyperthermophilic bacterium Thermotoga neapolitana, a member of the order Thermotogales. The CO2-activated pathway enables non-competitive synthesis of hydrogen and L-lactic acid at high yields, making it an economically attractive process for bioenergy production. In this work, we discovered and characterized CLF in Thermotoga sp. strain RQ7, a naturally competent strain, opening a new avenue for molecular investigation of the pathway. Evaluation of the fermentation products and expression analyses of key CLF-genes by RT-PCR revealed similar CLF-phenotypes between T. neapolitana and T. sp. strain RQ7, which were absent in the non-CLF-performing strain T. maritima. Key CLF enzymes, such as PFOR, HYD, LDH, RNF, and NFN, are up-regulated in the two CLF strains. Another important finding is the up-regulation of V-ATPase, which couples ATP hydrolysis to proton transport across the membranes, in the two CLF-performing strains. The fact that V-ATPase is absent in T. maritima suggested that this enzyme plays a key role in maintaining the necessary proton gradient to support high demand of reducing equivalents for simultaneous hydrogen and lactic acid synthesis in CLF.

Improvement of CO2 and Acetate Coupling into Lactic Acid by Genetic Manipulation of the Hyperthermophilic Bacterium Thermotoga neapolitana.

Capnophilic lactic fermentation (CLF) represents an attractive biotechnological process for biohydrogen production and synthesis of L-lactic acid from acetate and CO2. The present study focuses on a genetic manipulation approach of the Thermotoga neapolitana DSM33003 strain to enhance lactic acid synthesis by the heterologous expression of a thermostable acetyl-CoA synthetase that catalyses the irreversible acetate assimilation. Because of the scarcity of available genetic tools, each transformation step was optimized for T. neapolitana DSM33003 to cope with the specific needs of the host strain. Batch fermentations with and without an external source of acetate revealed a strongly increased lactate production (up to 2.5 g/L) for the recombinant strain compared to wild type. In the engineered bacterium, the assimilation of CO2 into lactic acid was increased 1.7 times but the hydrogen yield was impaired in comparison to the wild type strain. Analysis of fermentation yields revealed an impaired metabolism of hydrogen in the recombinant strain that should be addressed in future studies. These results offer an important prospective for the development of a sustainable approach that combines carbon capture, energy production from renewable source, and the synthesis of high value-added products, which will be addressed in future studies.

CO2-Induced Transcriptional Reorganization: Molecular Basis of Capnophillic Lactic Fermentation in Thermotoga neapolitana.

Capnophilic lactic fermentation (CLF) is a novel anaplerotic pathway able to convert sugars to lactic acid (LA) and hydrogen using CO2 as carbon enhancer in the hyperthermophilic bacterium Thermotoga neapolitana. In order to give further insights into CLF metabolic networks, we investigated the transcriptional modification induced by CO2 using a RNA-seq approach. Transcriptomic analysis revealed 1601 differentially expressed genes (DEGs) in an enriched CO2 atmosphere over a total of 1938 genes of the T. neapolitana genome. Transcription of PFOR and LDH genes belonging to the CLF pathway was up-regulated by CO2 together with 6-phosphogluconolactonase (6PGL) and 6-phosphogluconate dehydratase (EDD) of the Entner–Doudoroff (ED) pathway. The transcriptomic study also revealed up-regulation of genes coding for the flavin-based enzymes NADH-dependent reduced ferredoxin:NADP oxidoreductase (NFN) and NAD-ferredoxin oxidoreductase (RNF) that control supply of reduced ferredoxin and NADH and allow energy conservation-based sodium translocation through the cell membrane. These results support the hypothesis that CO2 induces rearrangement of the central carbon metabolism together with activation of mechanisms that increase availability of the reducing equivalents that are necessary to sustain CLF. In this view, this study reports a first rationale of the molecular basis of CLF in T. neapolitana and provides a list of target genes for the biotechnological implementation of this process.

Hydrogen and lactic acid synthesis by the wild-type and a laboratory strain of the hyperthermophilic bacterium Thermotoga neapolitana DSMZ 4359T under capnophilic lactic fermentation conditions

Thermotoga neapolitana is a hyperthermophilic eubacterium that produces hydrogen by sugar fermentation. A lab strain of T. neapolitana DSMZ 4359T maintained in a CO2-enriched atmosphere showed a stable increase of lactic acid production under capnophilic lactic fermentation (CLF) conditions. The genotypic comparison between the putative mutant (TN-CMut) and the original strain DSMZ 4359T (WT4359) revealed 88.1 (±2.4)% DNA homology. RiboPrint® and MALDI-TOF mass analyses support a genetic differentiation beyond subspecies level. The phenotypic characterization indicated a high correlation between the two strains, except for the lactic acid production. Under identical operating conditions, the lab mutant produced significantly more lactic acid than the parent strain without impairing the hydrogen yield. The highest divergence between TN-CMut and WT4359 was observed for fermentation of glucose or lactose at 80 °C. Based on these results, we propose that the lab strain is a new subspecies of the genus Thermotoga that is named T. neapolitana subsp. capnolactica with regards to its improved feature to produce lactic acid under capnophilic conditions.

Kinetic modeling of fermentative hydrogen production by Thermotoga neapolitana

A kinetic model of fermentative hydrogen (H2) production was developed for the hyperthermophilic marine bacterium Thermotoga neapolitana DSMZ 4359T using glucose as the substrate. The model was built based on anaerobic digestion model no.1 (ADM1) by IWA using Monod-like kinetic equations. Several series of pH controlled batch fermentation tests were performed under pure CO2 or N2 atmosphere in serum bottles to evaluate the growth kinetic parameters. The kinetic parameters were estimated by applying the non-linear least square method to Monod and Michaelis–Menten kinetic equations. The experimentally estimated value of k, kS, Y and kd were 0.839 h−1, 1.42 g/L, 0.1204 and 0.0043 h−1, respectively. The kinetic parameters were analyzed for sensitivities and subsequently calibrated and validated by a separate set of experimental results. The model was particularly effective in estimating the biomass growth, substrate consumption, and H2 production except for acetate and lactate production, due to capnophilic condition (under CO2 atmosphere).

Introducing capnophilic lactic fermentation in a combined dark-photo fermentation process: a route to unparalleled H2 yields.

Two-stage process based on photofermentation of dark fermentation effluents is widely recognized as the most effective method for biological production of hydrogen from organic substrates. Recently, it was described an alternative mechanism, named capnophilic lactic fermentation, for sugar fermentation by the hyperthermophilic bacterium Thermotoga neapolitana in CO2-rich atmosphere. Here, we report the first application of this novel process to two-stage biological production of hydrogen. The microbial system based on T. neapolitana DSM 4359(T) and Rhodopseudomonas palustris 42OL gave 9.4 mol of hydrogen per mole of glucose consumed during the anaerobic process, which is the best production yield so far reported for conventional two-stage batch cultivations. The improvement of hydrogen yield correlates with the increase in lactic production during capnophilic lactic fermentation and takes also advantage of the introduction of original conditions for culturing both microorganisms in minimal media based on diluted sea water. The use of CO2 during the first step of the combined process establishes a novel strategy for biohydrogen technology. Moreover, this study opens the way to cost reduction and use of salt-rich waste as feedstock.

Transgenic Expression of a Bacterial Thermophilic Amylase in the Chlamydomonas reinhardtii Chloroplast to Facilitate Algal Biofuel Production

This study investigated expression of a thermophilic amylase (arAmyBH) from hyperthermophilic bacterium Thermotoga neapolitana into Chlamydomonas reinhardtii chloroplast to facilitate in vivo starch hydrolysis for algal biofuel production. The expression and accumulation of the recombinant protein in the algal chloroplast were verified by Western blot analysis. The recombinant arAmyBH was purified using Ni-NTA purification system. The optimum pH and temperature of arAmyBH were 5.5 and 60 °C, respectively. The enzymatic hydrolysis of the algal starch demonstrated that with supplemental commercial alpha-amylase, the transformant completely converted starch into reducing sugars in vivo at an elevated temperature.

Structure of a novel α-amylase AmyB fromThermotoga neapolitanathat produces maltose from the nonreducing end of polysaccharides

An intracellular α-amylase, AmyB, has been cloned from the hyperthermophilic bacterium Thermotoga neapolitana. AmyB belongs to glycoside hydrolase family 13 and liberates maltose from diverse substrates, including starch, amylose, amylopectin and glycogen. The final product of AmyB is similar to that of typical maltogenic amylases, but AmyB cleaves maltose units from the nonreducing end, which is a unique property of this α-amylase. In this study, the crystal structure of AmyB from T. neapolitana has been determined at 2.4 Å resolution, revealing that the monomeric AmyB comprises domains A, B and C like other α-amylases, but with structural variations. In the structure, a wider active site and a putative extra sugar-binding site at the top of the active site were found. Subsequent biochemical results suggest that the extra sugar-binding site is suitable for recognizing the nonreducing end of the substrates, explaining the unique activity of this enzyme. These findings provide a structural basis for the ability of an α-amylase that has the common α-amylase structure to show a diverse substrate specificity.

Crystal structure and thermostability of a putative α-glucosidase from Thermotoga neapolitana

Glycoside hydrolase family 4 (GH4) represents an unusual group of glucosidases with a requirement for NAD +, Mn 2+, and reducing conditions. We found a putative α-glucosidase belonging to GH4 in hyperthermophilic Gram-negative bacterium Thermotoga neapolitana. In this study, we recombinantly expressed the putative α-glycosidase from T. neapolitana, and determined the crystal structure of the protein at a resolution of 2.0 Å in the presence of Mn 2+ but in the absence of NAD +. The structure showed the dimeric assembly and the Mn 2+ coordination that other GH4 enzymes share. In comparison, we observed structural changes in T. neapolitana α-glucosidase by the binding of NAD +, which also increased the thermostability. Numerous arginine-mediated salt-bridges were observed in the structure, and we confirmed that the salt bridges correlated with the thermostability of the proteins. Disruption of the salt bridge that linked N-terminal and C-terminal parts at the surface dramatically decreased the thermostability. A mutation that changed the internal salt bridge to a hydrogen bond also decreased the thermostability of the protein. This study will help us to understand the function of the putative glucosidase and the structural features that affect the thermostability of the protein.

Identification of an extracellular thermostable glycosyl hydrolase family 13 α-amylase from Thermotoga neapolitana

We cloned the gene for an extracellular α-amylase, AmyE, from the hyperthermophilic bacterium Thermotoga neapolitana and expressed it in Escherichia coli. The molecular mass of the enzyme was 92 kDa as a monomer. Maximum activity was observed at pH 6.5 and temperature 75°C and the enzyme was highly thermostable. AmyE hydrolyzed the typical substrates for α-amylase, including soluble starch, amylopectin, and maltooli-gosaccharides. The hydrolytic pattern of AmyE was similar to that of a typical α-amylase; however, unlike most of the calcium (Ca(2+))-dependent α-amylases, the activity of AmyE was unaffected by Ca(2+). The specific activities of AmyE towards various substrates indicated that the enzyme preferred maltooligosaccharides which have more than four glucose residues. AmyE could not hydrolyze maltose and maltotriose. When maltoheptaose was incubated with AmyE at the various time courses, the products consisting of maltose through maltopentaose was evenly formed indicating that the enzyme acts in an endo-fashion. The specific activity of AmyE (7.4 U/mg at 75° C, pH 6.5, with starch as the substrate) was extremely lower than that of other extracellular α-amylases, which indicates that AmyE may cooperate with other highly active extracellular α-amylases for the breakdown of the starch or α-glucans into maltose and maltotriose before transport into the cell in the members of Thermotoga sp.

Molecular cloning and biochemical characterization of a heat-stable type I pullulanase from Thermotoga neapolitana

The gene encoding a type I pullulanase from the hyperthermophilic anaerobic bacterium Thermotoga neapolitana (pulA) was cloned in Escherichia coli and sequenced. The pulA gene from T. neapolitana showed 91.5% pairwise amino acid identity with pulA from Thermotoga maritima and contained the four regions conserved in all amylolytic enzymes. pulA encodes a protein of 843 amino acids with a 19-residue signal peptide. The pulA gene was subcloned and overexpressed in E. coli under the control of the T7 promoter. The purified recombinant enzyme (rPulA) produced a 93-kDa protein with pullulanase activity. rPulA was optimally active at pH 5–7 and 80°C and had a half-life of 88min at 80°C. rPulA hydrolyzed pullulan, producing maltotriose, and hydrolytic activities were also detected with amylopectin, starch, and glycogen, but not with amylose. This substrate specificity is typical of a type I pullulanase. Thin layer chromatography of the reaction products in the reaction with pullulan and aesculin showed that the enzyme had transglycosylation activity. Analysis of the transfer product using NMR and isoamylase treatment revealed it to be α-maltotriosyl-(1,6)-aesculin, suggesting that the enzyme transferred the maltotriosyl residue of pullulan to aesculin by forming α-1,6-glucosidic linkages. Our findings suggest that the pullulanase from T. neapolitana is the first thermostable type I pullulanase which has α-1,6-transferring activity.

Characterization of an exo-acting intracellular α-amylase from the hyperthermophilic bacterium Thermotoga neapolitana

We cloned and expressed the gene for an intracellular alpha-amylase, designated AmyB, from the hyperthermophilic bacterium Thermotoga neapolitana in Escherichia coli. The putative intracellular amylolytic enzyme contained four regions that are highly conserved among glycoside hydrolase family (GH) 13 alpha-amylases. AmyB exhibited maximum activity at pH 6.5 and 75 degrees C, and its thermostability was slightly enhanced by Ca2+. However, Ca2+ was not required for the activity of AmyB as EDTA had no effect on enzyme activity. AmyB hydrolyzed the typical substrates for alpha-amylase, including soluble starch, amylose, amylopectin, and glycogen, to liberate maltose and minor amount of glucose. The hydrolytic pattern of AmyB is most similar to those of maltogenic amylases (EC 3.2.1.133) among GH 13 alpha-amylases; however, it can be distinguished by its inability to hydrolyze pullulan and beta-cyclodextrin. AmyB enzymatic activity was negligible when acarbose, a maltotetraose analog in which a maltose residue at the nonreducing end was replaced by acarviosine, was present, indicating that AmyB cleaves maltose units from the nonreducing end of maltooligosaccharides. These results indicate that AmyB is a new type exo-acting intracellular alpha-amylase possessing distinct characteristics that distinguish it from typical alpha-amylase and cyclodextrin-/pullulan-hydrolyzing enzymes.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of Thermotoga neapolitana beta-glucosidase B.

Beta-glucosidases belong to families 1, 3 and 9 of the glycoside hydrolases and act on cello-oligosaccharides. Family 1 and 3 enzymes are retaining and are reported to have transglycosylation activity, which can be used to produce oligosaccharides and glycoconjugates. Family 3 enzymes are less well characterized than their family 1 homologues and to date only two crystal structures have been solved. Here, the expression, purification, crystallization and X-ray diffraction data of a family 3 beta-glucosidase from the hyperthermophilic bacterium Thermotoga neapolitana are reported. Crystals of selenomethionine-substituted protein have also been grown. The crystals belong to space group C222(1), with unit-cell parameters a = 74.9, b = 127.0, c = 175.2 A. Native data have been collected to 2.4 A resolution and the structure has been solved to 2.7 A using the selenomethionine MAD method. Model building and refinement of the structure are under way.

Hyperthermophilic Thermotoga Arginine Repressor Binding to Full-length Cognate and Heterologous Arginine Operators and to Half-site Targets

The degree of sequence conservation of arginine repressor proteins (ArgR) and of the cognate operators (tandem pairs of 18 bp imperfect palindromes, ARG boxes) in evolutionarily distant bacteria is unusually high, and the global mechanism of ArgR-mediated regulation appears to be similar. However, here we demonstrate that the arginine repressor from the hyperthermophilic bacterium Thermotoga neapolitana (ArgRTn) exhibits characteristics that clearly distinguish this regulator from the well-studied homologues from Escherichia coli, Bacillus subtilis and B.stearothermophilus. A high-resolution contact map of ArgRTn binding to the operator of the biosynthetic argGHCJBD operon of Thermotoga maritima indicates that ArgRTn establishes all of its strong contacts with a single ARG box-like sequence of the operator only. Protein array and electrophoretic mobility-shift data demonstrate that ArgRTn has a remarkable capacity to bind to arginine operators from Gram-negative and Gram-positive bacteria, and to single ARG box-bearing targets. Moreover, the overall effect of l-arginine on the apparent Kd of ArgRTn binding to various cognate and heterologous operator fragments was minor with respect to that observed with diverse bacterial arginine repressors. We demonstrate that this unusual behaviour for an ArgR protein can, to a large extent, be ascribed to the presence of a serine residue at position 107 of ArgRTn, instead of the highly conserved glutamine that is involved in arginine binding in the E.coli repressor. Consistent with these results, ArRTn was found to behave as a superrepressor in E.coli, inhibiting growth in minimal medium, even supplemented with arginine, whereas similar constructs bearing the S107Q mutant allele did not inhibit growth. We assume that ArgRTn, owing to its broad target specificity and its ability to bind single ARG box sequences, might play a more general regulatory role in Thermotoga

F- and V-type ATPases in the hyperthermophilic bacterium Thermotoga neapolitana.

Two gene clusters encoding F- or V-type ATPases were found in genomic DNA of the hyperthermophilic bacterium Thermotoga neapolitana. The subunit genes of each ATPase formed an operon. While the gene arrangement in the operon of the F-type ATPase resembled those in eukaryotic organelles and bacteria, that of the V-type ATPase was different from those reported for archaea, bacteria, or eukaryotes. Both ATPases were found to be expressed in the cells of T. neapolitana by Western blot analysis. Although V-type ATPase could not be rendered soluble, F-type ATPase was solubilized with 1% Triton X-100 and characterized. This is the first report of the coexistence of both F- and V-type ATPases in hyperthermophilic bacteria. It has recently been shown by a genome analysis that Thermotoga maritima has no V-type ATPase gene cluster but does have an F-type ATPase gene cluster; however, part of a gene for the D-subunit of the V-type ATPase gene has been reported in the T. maritima genome. Evolution of the two types of ATPases in Thermotoga is discussed.

Characterization of extremely thermostable enzymatic breakers (α-1,6-galactosidase and β-1,4-mannanase) from the hyperthermophilic bacterium Thermotoga neapolitana 5068 for hydrolysis of guar gum

An alpha-galactosidase and a beta-mannanase produced by the hyperthermophilic bacterium, Thermotoga neapolitana 5068 (TN5068), separately and together, were evaluated for their ability to hydrolyze guar gum in relation to viscosity reduction of guar-based hydraulic fracturing fluids used in oil and gas well stimulation. In such applications, premature guar gum hydrolysis at lower temperatures before the fracturing process is completed is undesirable, whereas thermostability and thermoactivity are advantageous. Hyperthermophilic enzymes presumably possess both characteristics. The purified alpha-galactosidase was found to have a temperature optimum of 100-105 degrees C with a half-life of 130 minutes at 90 degrees C and 3 min at 100 degrees C, while the purified beta-mannanase was found to have a temperature optimum of 91 degrees C and a half-life of 13h at this temperature and 35 min at 100 degrees C.These represent the most thermostable versions of these enzymes yet reported. At 25 degrees C, TN5068 culture supernatants, containing the two enzyme activities, reduced viscosity of a 0.7% (wt) guar gum solution by a factor of 1.4 after a 1.5-h incubation period and by a factor of 2.4 after 5 h. This is in contrast to a viscosity reduction of 100-fold after 1.5 h and 375-fold after 5 h for a commercial preparation of these enzymes from Aspergillus niger. In contrast, at 85 degrees C, the TN5068 enzymes reduced viscosity by 30-fold after 1.5 h and 100-fold after 5 h compared to a 2.5-fold reduction after 5 h for the control. The A. niger enzymes were less effective at 85 degrees C (1.6-fold reduction after 1.5 h and a 4.2-fold reduction after 5 h), presumably due to their thermal lability at this temperature. Furthermore, it was determined that the purified beta-mannanase alone can substantially reduce viscosity of guar solutions, while the alpha-galactosidase alone had limited viscosity reduction activity. However, the alpha-galactosidase appeared to minimize residual particulate matter when used in conjunction with the beta-mannanase. This could be the result of extensive hydrolysis of the alpha-1,6 linkages between mannose and galactose units in guar, allowing more extensive hydrolysis of the mannan chain by the beta-mannanase. The use of thermostable enzymatic breakers from hyperthermophiles in hydraulic fracturing could be used to improve well stimulation and oil and gas recovery.

Thermostability, oligomerization and DNA-binding properties of the regulatory protein ArgR from the hyperthermophilic bacterium Thermotoga neapolitana.

The hexameric regulatory protein ArgR formed by arginine-mediated dimerization of identical trimers governs the expression of genes required for arginine metabolism and some other genes in mesophilic and moderately thermophilic bacteria. We have cloned the argR gene from two hyperthermophilic bacteria of the genus Thermotoga. The two-domain ArgR proteins encoded by T. neapolitana and T. maritima share a low degree of sequence similarity with other bacterial arginine repressors. The ArgR protein from T. neapolitana binds to an operator located just upstream of its coding sequence and, therefore, the argR gene may be autoregulated. The protein has extremely high intrinsic thermostability and tolerance to urea. Moreover, its binding to target DNA increases the melting temperature by approximately 15 degrees C. The formation of oligomeric ArgR-DNA complexes is a function of protein concentration, with hexameric complexes being favoured at higher concentrations. In the presence of arginine the hyperthermophilic ArgR protein binds to its own operator, argRo, only by forming hexamer ArgR-DNA complexes, whereas both trimer-DNA and hexamer-DNA complexes are detected in the absence of arginine. However, the affinity of T. neapolitana ArgR for DNA has been found to be higher for a mixture of trimers and non-bound hexamers than for arginine-bound hexamers. Our data indicate that genes for arginine biosynthesis are clustered in a putative operon, which could also be regulated by the ArgR protein, in the hyperthermophilic host.

Membrane-associated redox activities in Thermotoga neapolitana.

Elemental sulfur reduction by the hyperthermophilic bacterium Thermotoga neapolitana provides an alternative to hydrogen evolution during fermentation. Electrons are transferred from reduced cofactors (ferredoxin and NADH) to sulfur by a series of unknown steps. One enzyme that may be involved is an NADH:methyl viologen oxidoreductase (NMOR), an activity that in other fermenting organisms is associated with NADH:ferredoxin oxidoreductase. We found that 83% of NMOR activity was contained in the pellet fraction of cell extracts subjected to ultracentrifugation. This pellet fraction, presumably containing cell membranes, was required for electron transfer to NAD+ from ferredoxin-dependent pyruvate oxidation. However, the NMOR activity in this fraction used neither Thermotoga nor clostridial ferredoxins as substrates. NMOR activity was also detected in aerobically prepared vesicles. By comparison with ATPase activities, NMOR was found primarily on the cytoplasmic face of these vesicles. During these studies, an extracytoplasmic hydrogenase activity was discovered. In contrast to the soluble hydrogenase, this hydrogenase activity was completely inhibited when intact cells were treated with cupric chloride and was present on the extracytoplasmic face of vescides. In contrast to a soluble hydrogenase reported in Thermotoga maritima, this activity was air-stable and was inhibited by low concentrations of nitrite.

Coregulation of beta-galactoside uptake and hydrolysis by the hyperthermophilic bacterium Thermotoga neapolitana

Regulation of the beta-galactoside transport system in response to growth substrates in the extremely thermophilic anaerobic bacterium Thermotoga neapolitana was studied with the nonmetabolizable analog methyl-beta-D-thiogalactopyranoside (TMG) as the transport substrate. T. neapolitana cells grown on galactose or lactose accumulated TMG against a concentration gradient in an intracellular free sugar pool that was exchangeable with external galactose or lactose and showed induced levels of beta-galactosidase. Cells grown on glucose, maltose, or galactose plus glucose showed no capacity to accumulate TMG, though these cells carried out active transport of the nonmetabolizable glucose analog 2-deoxy-D-glucose. Glucose neither inhibited TMG uptake nor caused efflux of preaccumulated TMG; rather, glucose promoted TMG uptake by supplying metabolic energy. These data show that beta-D-galactosides are taken up by T. neapolitana via an active transport system that can be induced by galactose or lactose and repressed by glucose but which is not inhibited by glucose. Thus, the phenomenon of catabolite repression is present in T. neapolitana with respect to systems catalyzing both the transport and hydrolysis of beta-D-galactosides, but inducer exclusion and inducer expulsion, mechanisms that regulate permease activity, are not present. Regulation is manifest at the level of synthesis of the beta-galactoside transport system but not in the activity of the system.

The multidomain xylanase A of the hyperthermophilic bacterium Thermotoga neapolitana is extremely thermoresistant.

The nucleotide sequence of the xynA gene, encoding extracellular xylanase A of Thermotoga neapolitana, was determined. The xynA gene was 3264 base pairs (bp) long and encoded a putative polypeptide of 1055 amino acids. Three different domains were identified by sequence comparison and functional analysis of proteins with N- and/or C-terminal deletions. The core domain displayed significant homology to members of the glycosyl hydrolase family 10. N- and C-terminal domains were dispensable for enzymatic activity and seemed to be responsible for thermostability and cellulose binding, respectively. The intact gene and its truncated variants were expressed in Escherichia coli and purified for biochemical characterization. The enzyme was shown to act as an endo-1,4-beta-xylanase, but minor activities against lichenan, barley glucan, methylumbelliferyl cellobioside and p-nitrophenyl xyloside were also detected. The specific activity and pH and temperature optima for hydrolysis of oat xylan were 111.3 U.mg-1, 5.5 and 102 degrees C, respectively. The endoxylanase was stable at 90 degrees C and retained 50% activity when incubated for 2 h at 100 degrees C.