Characterization of Di-myo-Inositol-1,1(prm1)-Phosphate in the Hyperthermophilic Bacterium Thermotoga maritima

Di-myo-inositol-1,1(prm1)-phosphate (DIP) is present at a significant concentration ((symbl)160 nmol/mg of protein) in the cytoplasm of the hyperthermophilic bacterium Thermotoga maritima. The concentration of DIP was independent of the pH of the growth medium or the cell growth phase but increased with increasing concentrations of NaCl in the growth medium, reaching a maximum ((symbl)450 nmol/mg of protein) at 0.4 to 0.6 M NaCl. A large-scale purification procedure for DIP which yields approximately 18 g of DIP per kg of cells (wet weight) is described. Purified DIP was stable at 90(deg)C for at least 5 h. The presence of DIP (50 mM) did not increase the stability at 90(deg)C of pure forms of the hydrogenase or pyruvate ferredoxin oxidoreductase of T. maritima, suggesting that DIP is not a general thermoprotectant.     

Two Extremely Thermostable Xylanases of the Hyperthermophilic Bacterium Thermotoga maritima MSB8

During growth with xylose or xylan as the source of carbon, xylanase production by Thermotoga maritima MSB8 was enhanced about 10-fold compared with growth with glucose or starch. Two extremely thermostable endoxylanases (1,4-(beta)-d-xylan-xylanohydrolase, EC 3.2.1.8), designated XynA and XynB, were identified and purified from cells of this organism. XynA and XynB occurred as proteins with apparent molecular masses of about 120 and 40 kDa, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Maximum activity at the optimal pH (pH 6.2 and pH 5.4 for XynA and XynB, respectively) was measured at about 92(deg)C for XynA (10-min assay) and at about 105(deg)C for XynB (5-min assay). XynB activity was stimulated twofold by the addition of 500 mM NaCl, while XynA displayed maximum activity without the addition of salt. Both xylanases were tolerant of relatively high salt concentrations. At 2 M (about 12% wt/vol) NaCl, XynA and XynB retained 49 and 65%, respectively, of their maximum activities. In contrast to XynB, XynA was able to adsorb to microcrystalline cellulose. Antibodies raised against a recombinant truncated XynA protein cross-reacted with XynB, indicating that the enzymes may have sequence or structural similarities. Part of the xylanase activity appeared to be associated with the outer membrane of T. maritima cells, since more than 40% of the total xylanase activity present in the crude cellular extract was found in the membrane fraction after high-speed centrifugation. Most of the membrane-bound activity appeared to be due to the 120-kDa xylanase XynA.     

Characterization of a Cellobiose Phosphorylase from a Hyperthermophilic Eubacterium,Thermotoga maritima MSB8

The cepA putative gene encoding a cellobiose phosphorylase of Thermotoga maritima MSB8 was cloned, expressed in Escherichia coli BL21-codonplus-RIL and characterized in detail. The maximal enzyme activity was observed at pH 6.2 and 80°C. The energy of activation was 74 kJ/mol. The enzyme was stable for 30 min at 70°C in the pH range of 6-8. The enzyme phosphorolyzed cellobiose in an random-ordered bi bi mechanism with the random binding of cellobiose and phosphate followed by the ordered release of D-glucose and α-D-glucose-1-phosphate. The K _m for cellobiose and phosphate were 0.29 and 0.15 mM respectively, and the k _cat was 5.4 s^-1. In the synthetic reaction, D-glucose, D-mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, and 6-deoxy-D-glucose were found to act as glucosyl acceptors. Methyl-β-D-glucoside also acted as a substrate for the enzyme and is reported here for the first time as a substrate for cellobiose phosphorylases. D-Xylose had the highest (40 s^-1) k _cat followed by 6-deoxy-D-glucose (17 s^-1) and 2-deoxy-D-glucose (16 s^-1). The natural substrate, D-glucose with the k _cat of 8.0 s^-1 had the highest (1.1×10⁴ M^-1 s^-1) k _cat/K _m compared with other glucosyl acceptors. D-Glucose, a substrate of cellobiose phosphorylase, acted as a competitive inhibitor of the other substrate, α-D-glucose-1-phosphate, at higher concentrations.     

Identification and Characterization of a Novel Intracellular Alkaline α-Amylase from the Hyperthermophilic Bacterium Thermotoga maritima MSB8

The gene for a novel α-amylase, designated AmyC, from the hyperthermophilic bacterium Thermotoga maritima was cloned and heterologously overexpressed in Escherichia coli. The putative intracellular enzyme had no amino acid sequence similarity to glycoside hydrolase family (GHF) 13 α-amylases, yet the range of substrate hydrolysis and the product profile clearly define the protein as an α-amylase. Based on sequence similarity AmyC belongs to a subgroup within GHF 57. On the basis of amino acid sequence similarity, Glu185 and Asp349 could be identified as the catalytic residues of AmyC. Using a 60-min assay, the maximum hydrolytic activity of the purified enzyme, which was dithiothreitol dependent, was found to be at 90°C. AmyC displayed a remarkably high pH optimum of pH 8.5 and an unusual sensitivity towards both ATP and EDTA.     

Regulation of Endo-Acting Glycosyl Hydrolases in the Hyperthermophilic Bacterium Thermotoga maritima Grown on Glucan- and Mannan-Based Polysaccharides

The genome sequence of the hyperthermophilic bacterium Thermotoga maritima encodes a number of glycosyl hydrolases. Many of these enzymes have been shown in vitro to degrade specific glycosides that presumably serve as carbon and energy sources for the organism. However, because of the broad substrate specificity of many glycosyl hydrolases, it is difficult to determine the physiological substrate preferences for specific enzymes from biochemical information. In this study, T. maritima was grown on a range of polysaccharides, including barley β-glucan, carboxymethyl cellulose, carob galactomannan, konjac glucomannan, and potato starch. In all cases, significant growth was observed, and cell densities reached 10⁹ cells/ml. Northern blot analyses revealed different substrate-dependent expression patterns for genes encoding the various endo-acting β-glycosidases; these patterns ranged from strong expression to no expression under the conditions tested. For example, cel74 (TM0305), a gene encoding a putative β-specific endoglucananse, was strongly expressed on all substrates tested, including starch, while no evidence of expression was observed on any substrate for lam16 (TM0024), xyl10A (TM0061), xyl10B (TM0070), and cel12A (TM1524), which are genes that encode a laminarinase, two xylanases, and an endoglucanase, respectively. The cel12B (TM1525) gene, which encodes an endoglucanase, was expressed only on carboxymethyl cellulose. An extracellular mannanase encoded by man5 (TM1227) was expressed on carob galactomannan and konjac glucomannan and to a lesser extent on carboxymethyl cellulose. An unexpected result was the finding that the cel5A (TM1751) and cel5B (TM1752) genes, which encode putative intracellular, β-specific endoglucanases, were induced only when T. maritima was grown on konjac glucomannan. To investigate the biochemical basis of this finding, the recombinant forms of Man5 (M_r, 76,900) and Cel5A (M_r, 37,400) were expressed in Escherichia coli and characterized. Man5, a T. maritima extracellular enzyme, had a melting temperature of 99°C and an optimun temperature of 90°C, compared to 90 and 80°C, respectively, for the intracellular enzyme Cel5A. While Man5 hydrolyzed both galactomannan and glucomannan, no activity was detected on glucans or xylans. Cel5A, however, not only hydrolyzed barley β-glucan, carboxymethyl cellulose, xyloglucan, and lichenin but also had activity comparable to that of Man5 on galactomannan and higher activity than Man5 on glucomannan. The biochemical characteristics of Cel5A, the fact that Cel5A was induced only when T. maritima was grown on glucomannan, and the intracellular localization of Cel5A suggest that the physiological role of this enzyme includes hydrolysis of glucomannan oligosaccharides that are transported following initial hydrolysis by extracellular glycosidases, such as Man5.     

Characterization of a thermostable recombinant beta-galactosidase from Thermotoga maritima

AIMS: Characterization of a thermostable recombinant beta-galactosidase from Thermotoga maritima for the hydrolysis of lactose and the production of galacto-oligosaccharides. METHODS AND RESULTS: A putative beta-galactosidase gene of Thermotoga maritima was expressed in Escherichia coli as a carboxyl terminal His-tagged recombinant enzyme. The gene encoded a 1100-amino acid protein with a calculated molecular weight of 129,501. The expressed enzyme was purified by heat treatment, His-tag affinity chromatography, and gel filtration. The optimum temperatures for beta-galactosidase activity were 85 and 80 degrees C with oNPG and lactose, respectively. The optimum pH value was 6.5 for both oNPG and lactose. In thermostability experiments, the enzyme followed first-order kinetics of thermal inactivation and its half-life times at 80 and 90 degrees C were 16 h and 16 min, respectively. Mn2+ was the most effective divalent cation for beta-galactosidase activity on both oNPG and lactose. The Km and Vmax values of the thermostable enzyme for oNPG at 80 degrees C were 0.33 mm and 79.6 micromol oNP min(-1) mg(-1). For lactose, the Km and Vmax values were dependent on substrate concentrations; 1.6 and 63.3 at lower concentrations up to 10 mm of lactose and 27.8 mm and 139 micromol glucose min(-1) mg(-1) at higher concentrations, respectively. The enzyme displayed non-Michaelis-Menten reaction kinetics with substrate activation, which was explained by simultaneous reactions of hydrolysis and transgalactosylation. CONCLUSIONS: The results suggest that the thermostable enzyme may be suitable for both the hydrolysis of lactose and the production of galacto-oligosaccharides. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work contribute to the knowledge of hydrolysis and transgalactosylation performed by beta-galactosidase of hyperthermophilic bacteria.     

Characterization of RNase P from Thermotoga maritima

The protein subunit of RNase P from a thermophilic bacterium, Thermotoga maritima, was overexpressed in and purified from Escherichia coli. The cloned protein was reconstituted with the RNA subunit transcribed in vitro. The temperature optimum of the holoenzyme is near 50°C, with no enzymatic activity at 65°C or above. This finding is in sharp contrast to the optimal growth temperature of T.maritima, which is near 80°C. However, in heterologous reconstitution experiments in vitro with RNase P subunits from other species, we found that the protein subunit from T.maritima was responsible for the comparative thermal stability of such complexes.     

Characterization of acetohydroxyacid synthase from the hyperthermophilic bacterium Thermotoga maritima

Acetohydroxyacid synthase (AHAS) is the key enzyme in branched chain amino acid biosynthesis pathway. The enzyme activity and properties of a highly thermostable AHAS from the hyperthermophilic bacterium Thermotoga maritima is being reported. The catalytic and regulatory subunits of AHAS from T. maritima were over-expressed in Escherichia coli. The recombinant subunits were purified using a simplified procedure including a heat-treatment step followed by chromatography. A discontinuous colorimetric assay method was optimized and used to determine the kinetic parameters. AHAS activity was determined to be present in several Thermotogales including T. maritima. The catalytic subunit of T. maritima AHAS was purified approximately 30-fold, with an AHAS activity of approximately 160±27 U/mg and native molecular mass of 156±6 kDa. The regulatory subunit was purified to homogeneity and showed no catalytic activity as expected. The optimum pH and temperature for AHAS activity were 7.0 and 85 °C, respectively. The apparent K_m and V_max for pyruvate were 16.4±2 mM and 246±7 U/mg, respectively. Reconstitution of the catalytic and regulatory subunits led to increased AHAS activity. This is the first report on characterization of an isoleucine, leucine, and valine operon (ilv operon) enzyme from a hyperthermophilic microorganism and may contribute to our understanding of the physiological pathways in Thermotogales. The enzyme represents the most active and thermostable AHAS reported so far.     

Characterization of a thermostable endo-beta-1,4-D-galactanase from the hyperthermophile Thermotoga maritima

A putative endo-beta-1,4-D-galactanase gene of Thermotoga maritima was cloned and overexpressed in Escherichia coli. The recombinant enzyme hydrolyzed pectic galactans and produced D-galactose, beta-1,4-D-galactobiose, beta-1,4-D-galactotriose, and beta-1,4-D-galactotetraose. The enzyme displayed optimum activity at 90 degrees C and pH 7.0. It was slowly inactivated above pH 8.0 and below pH 5.0 and stable at temperatures up to 80 degrees C.     

Characterization of a tetrameric inositol monophosphatase from the hyperthermophilic bacterium Thermotoga maritima.

Inositol monophosphatase (I-1-Pase) catalyzes the dephosphorylation step in the de novo biosynthetic pathway of inositol and is crucial for all inositol-dependent processes. An extremely heat-stable tetrameric form of I-1-Pase from the hyperthermophilic bacterium Thermotoga maritima was overexpressed in Escherichia coli. In addition to its different quaternary structure (all other known I-1-Pases are dimers), this enzyme displayed a 20-fold higher rate of hydrolysis of D-inositol 1-phosphate than of the L isomer. The homogeneous recombinant T. maritima I-1-Pase (containing 256 amino acids with a subunit molecular mass of 28 kDa) possessed an unusually high V(max) (442 micromol min(-1) mg(-1)) that was much higher than the V(max) of the same enzyme from another hyperthermophile, Methanococcus jannaschii. Although T. maritima is a eubacterium, its I-1-Pase is more similar to archaeal I-1-Pases than to the other known bacterial or mammalian I-1-Pases with respect to substrate specificity, Li(+) inhibition, inhibition by high Mg(2+) concentrations, metal ion activation, heat stability, and activation energy. Possible reasons for the observed kinetic differences are discussed based on an active site sequence alignment of the human and T. maritima I-1-Pases.     

极耐热性阿拉伯糖苷酶基因的表达、纯化及酶学性质研究

采用PCR从海栖热袍菌(Thermotoga maritima)克隆出编码极耐热稳定性阿拉伯糖苷酶基因,以pET20b为表达质粒,与其C末端6个组氨酸标签序列融合,在大肠杆菌中得到高效表达。基因表达产物通过热处理和亲和层析柱纯化后,酶纯度达电泳均一。纯化重组酶稳定性检测表明,阿拉伯糖苷酶活性最适作用温度和最适作用pH分别为90～95℃和pH 5.0～5.5,在pH 4.2～8.2之间酶活力稳定,95℃的半衰期为4h;SDSPAGE测得酶的分子量为56.57 kD,与理论推算值相吻合。在所测定的底物中,阿拉伯糖苷酶仅对对硝基苯阿拉伯呋喃糖苷（pNPAF）有专一性水解作用,其动力学参数Km值为018mmol/L, Vmax为139μmol/min·mg。     

Dihydrodipicolinate synthase from Thermotoga maritima

DHDPS (dihydrodipicolinate synthase) catalyses the branch point in lysine biosynthesis in bacteria and plants and is feedback inhibited by lysine. DHDPS from the thermophilic bacterium Thermotoga maritima shows a high level of heat and chemical stability. When incubated at 90 degrees C or in 8 M urea, the enzyme showed little or no loss of activity, unlike the Escherichia coli enzyme. The active site is very similar to that of the E. coli enzyme, and at mesophilic temperatures the two enzymes have similar kinetic constants. Like other forms of the enzyme, T. maritima DHDPS is a tetramer in solution, with a sedimentation coefficient of 7.2 S and molar mass of 133 kDa. However, the residues involved in the interface between different subunits in the tetramer differ from those of E. coli and include two cysteine residues poised to form a disulfide bond. Thus the increased heat and chemical stability of the T. maritima DHDPS enzyme is, at least in part, explained by an increased number of inter-subunit contacts. Unlike the plant or E. coli enzyme, the thermophilic DHDPS enzyme is not inhibited by (S)-lysine, suggesting that feedback control of the lysine biosynthetic pathway evolved later in the bacterial lineage.     

海栖热袍菌葡萄糖异构酶基因xylA的克隆、表达及酶学性质

海栖热袍菌(Thermotoga maritima)是嗜极端高温的厌氧细菌,其产生的葡萄糖异构酶由于其出色的耐热性有着潜在的工业应用价值.由于海栖热袍菌苛刻的培养条件导致其葡萄糖异构酶产量较低.通过PCR方法克隆编码T. maritima MSB8葡萄糖异构酶基因xylA,构建重组质粒pHsh-xylA,转入Escherichia coli JM109,通过热激诱导表达.通过热处理和离子交换层析纯化两步得到电泳纯的酶制品,纯化倍数和回收率分别为8.02和49.02.对酶学性质研究表明,该重组酶为金属离子激活性酶,Mg2 ,Co2 对相对酶活有很强的激活作用,其最适pH为7.0,最适反应温度为95℃,且在pH 6～8之间有着较好的稳定性,在95℃下半衰期长达5 h以上.以葡萄糖为底物时的表观Km和Vmax分别为105 mmol/L和45.2 mol/min·mg.     

Characterization of a cellobiose phosphorylase from a hyperthermophilic eubacterium,Thermotoga maritima MSB8

The cepA putative gene encoding a cellobiose phosphorylase of Thermotoga maritima MSB8 was cloned, expressed in Escherichia coli BL21-codonplus-RIL and characterized in detail. The maximal enzyme activity was observed at pH 6.2 and 80 degrees C. The energy of activation was 74 kJ/mol. The enzyme was stable for 30 min at 70 degrees C in the pH range of 6-8. The enzyme phosphorolyzed cellobiose in an random-ordered bi bi mechanism with the random binding of cellobiose and phosphate followed by the ordered release of D-glucose and alpha-D-glucose-1-phosphate. The Km for cellobiose and phosphate were 0.29 and 0.15 mM respectively, and the kcat was 5.4 s(-1). In the synthetic reaction, D-glucose, D-mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, and 6-deoxy-D-glucose were found to act as glucosyl acceptors. Methyl-beta-D-glucoside also acted as a substrate for the enzyme and is reported here for the first time as a substrate for cellobiose phosphorylases. D-Xylose had the highest (40 s(-1)) kcat followed by 6-deoxy-D-glucose (17 s(-1)) and 2-deoxy-D-glucose (16 s(-1)). The natural substrate, D-glucose with the kcat of 8.0 s(-1) had the highest (1.1 x 10(4) M(-1) s(-1)) kcat/Km compared with other glucosyl acceptors. D-Glucose, a substrate of cellobiose phosphorylase, acted as a competitive inhibitor of the other substrate, alpha-D-glucose-1-phosphate, at higher concentrations.     

极耐热性b-葡萄糖醛酸酶的高效表达和酶学性质及其 应用

从海栖热袍菌克隆出编码热稳定性b-葡萄糖醛酸酶基因, 以热激载体pHsh为表达质粒, 在大肠杆菌中得到高效表达。基因表达产物通过一步热处理后, 酶纯度达电泳均一。纯化重组酶酶学性质研究表明, b-葡萄糖醛酸酶的最适反应温度为80oC, 最适反应pH为5.0, pH 5.8~ 8.2之间酶的稳定性较好, 80oC的半衰期为2 h, SDS-PAGE结果显示分子量为65.9 kD, 与理论推算值相吻合。以对硝基苯-b-葡萄糖醛酸苷(pNPG)为底物时, 其动力学参数Km值0.18 mmol/L, Vmax值为312 u/mg。初步的应用分析表明, 该重组酶能催化甘草酸转化为甘草次酸。