Purification and some properties of an extracellular protease (caldolysin) from an extreme thermophile

An extracellular metal-chelator-sensitive lytic protease (assigned the trivial name caldolysin) was isolated from a Thermus-like organism, Thermus T-351. Caldolysin was purified by affinity chromatography on Cbz-D-phenylalanine-TETA-Sepharose 4B and by gel filtration. It contained 13% carbohydrate, a single zinc atom, had a molecular weight of approx. 21,000, a pH optimum of 8 (azocasein substrate), and an isoelectric point of about 8.5. It was capable of hydrolysing many soluble and insoluble protein substrates, including collagen and elastin. No esterase activity was detected, and small peptides (less than four amino acids) and low molecular weight chromogenic substrates were not hydrolysed. A specificity for small aliphatic amino acids on either side of the splitting point was indicated. Caldolysin lysed heat-killed Gram-negative bacterial cells, but had little effect on Gram-positive organisms. Caldolysin exhibited a very high degree of thermostability (t 1/2(80 degrees C) approximately 30 h, t 1/2(90 degrees C) = 1 h). The stability (but not activity) was shown to be dependent on the presence of Ca2+ (t 1/2(75 degrees C, 10 mM calcium) greater than 193 h; t 1/2(75 degrees C, no calcium) = 4.8 min). None of the other metal ions tested (Co, Zn, Sr, Mg, Ba and Cu) was as effective as calcium in conferring thermostability of EDTA-treated caldolysin. Caldolysin was stable at room temperature in moderately acid and alkaline (pH 5 to 11) buffers for periods of greater than 90 days. Little loss of enzyme activity was detected after the incubation of caldolysin at 18 degrees C in the presence of 8 M urea, 6 M guanidine hydrochloride or 1% sodium dodecyl sulphate for 24 h. At 75 degrees C, the activity half-life of caldolysin in these denaturing agents was reduced to approx. 1 h, 1 h and more than 5 h, respectively.     

Analysis of a multicomponent thermostable DNA polymerase III replicase from an extreme thermophile

This report takes a proteomic/genomic approach to characterize the DNA polymerase III replication apparatus of the extreme thermophile, Aquifex aeolicus. Genes (dnaX, holA, and holB) encoding the subunits required for clamp loading activity (tau, delta, and delta') were identified. The dnaX gene produces only the full-length product, tau, and therefore differs from Escherichia coli dnaX that produces two proteins (gamma and tau). Nonetheless, the A. aeolicus proteins form a taudeltadelta' complex. The dnaN gene encoding the beta clamp was identified, and the taudeltadelta' complex is active in loading beta onto DNA. A. aeolicus contains one dnaE homologue, encoding the alpha subunit of DNA polymerase III. Like E. coli, A. aeolicus alpha and tau interact, although the interaction is not as tight as the alpha-tau contact in E. coli. In addition, the A. aeolicus homologue to dnaQ, encoding the epsilon proofreading 3'-5'-exonuclease, interacts with alpha but does not form a stable alpha.epsilon complex, suggesting a need for a brace or bridging protein to tightly couple the polymerase and exonuclease in this system. Despite these differences to the E. coli system, the A. aeolicus proteins function to yield a robust replicase that retains significant activity at 90 degrees C. Similarities and differences between the A. aeolicus and E. coli pol III systems are discussed, as is application of thermostable pol III to biotechnology.     

The cellulase activity of an extreme thermophile

Summary The carboxymethylcellulase activity concentrated from the extremely thermophilic anaerobe H173 was found to have a pH optimum of 6.5–7.0. The enzyme activity was stabilised by the addition of dithiothreitol and CaCl₂·2H₂O and was very stable at 80° C, retaining 77% of the initial activity after 120 min incubtation. At min and after 120 min only 3% of the initial activity remained. With the enzyme dissolved in buffer, glucose and cellobiose were formed from the hydrolosis of Avicel. In culture medium the Avicel-solubilising activity was insensitive to the presence of up tp 50 mm glucose and showed linear glucose accumulation over a period of days at 70° C. HPLC analysis established that glucose was the major end-product of hydrolysis in the culture broths.Offprint requests to: H. W. Morgan     

Aminoacyl-tRNA synthetases from an extreme thermophile, Thermus thermophilus HB8

Thermostable aminoacyl-tRNA synthetases specific to Val, Ile, Met and Glu were purified from an extreme thermophile, Thermus thermophilus HB8. As for the subunit compositions and molecular weights, these four aminoacyl-tRNA synthetases are similar to the corresponding enzymes from E. coli and B. stearothermophilus. Val-tRNA, Ile-tRNA and Met-tRNA synthetases from T. thermophilus have two tightly bound zinc ions, whereas Glu-tRNA synthetase does not. The amino acid compositions and secondary structures of Val-tRNA, Ile-tRNA and Met-tRNA synthetases are quite similar to one another. The conformational transition involving the anticodon of E. coli tRNAGlu as complexed with Glu-tRNA synthetase from T. thermophilus is necessary for the aminoacylation activity.     

Biochemical and molecular characterization of recombinant acidic and thermostable raw-starch hydrolysing α-amylase from an extreme thermophile Geobacillus thermoleovorans

A gene encoding acidic, thermostable and raw starch hydrolysing α-amylase was cloned from an extreme thermophile Geobacillus thermoleovorans and expressed. The ORF of 1650 bp encodes a 515 amino acid protein (Gt-amy) with a signal peptide of 34 amino acids at the N-terminus. Seven conserved sequences of GH-13 family have been found in its sequence. The specific enzyme activity of recombinant Gt-amy is 1723 U mg⁻¹ protein with a molecular mass of 59 kDa. It is optimally active at pH 5.0 and 80 °C with t_1/2 values of 283, 184 and 56 min at 70, 80 and 90 °C, respectively. The activation energy required for its temperature deactivation is 84.96 kJ mol⁻¹. Ca²⁺ strongly inhibits Gt-amy at 10 mM concentration, and inhibition kinetics with Ca²⁺ reveals that inhibition occurs as a result of binding to a lower affinity secondary Ca²⁺ binding site in the active centre in a mixed-type inhibition manner. The K_m and k_cat of the Gt-amy are 0.315 mg mL⁻¹ and 2.62 × 10³ s⁻¹, respectively. Gt-amy is Ca²⁺-independent at the concentration used in industrial starch saccharification, and hydrolyses raw corn and wheat starches efficiently, and thus, is applicable in starch saccharification at the industrial sub-gelatinization temperatures.     

Two glutamine synthetases from Bacillus caldolyticus, an extreme thermophile. Isolation, physicochemical and kinetic properties

Two distinctly different glutamine synthetase enzymes (EI and EII) have been isolated from the extreme thermophile Bacillus caldolyticus, grown on chemically defined medium at 70 degrees C. Purification to homogeneity mainly involves affinity chromatography and heat treatment with substrate protection. Biosynthesis of total enzyme activity can be repressed by at least 8-fold by high ammonia, with synthesis of EI being repressed more strongly than EII. A variety of chemical and biochemical tests failed to provide evidence for regulation of EI or EII by covalent modification, e.g. proteolysis, phosphorylation, or adenylylation. Neither of the thermophiic enzymes will cross-react with antibodies for the Escherichia coli or Bacillus subtilis glutamine synthetases. Both enzymes are composed of 12 subunits, each approximately 51,000 daltons. However, EI and EII differ significantly in their amino acid composition, isoelectric points (5.2 and 5.5, respectively), rates of migration on polyacrylamide electrophoresis gels at pH 6.8, and kinetic properties, EI is more active with Mg(II) than with Mn(II), but EII is more active with Mn(II) than Mg(II). Cd(II) activates EII more than EI, and only EI shows activity with Co(II). For both enzymes, the Mn(II)-stimulated activity is optimal at pH 6.0 to 6.5, with Mn(II)/ATP = 1.0, but the pH optimum with Mg(II) is near pH 7.5, however, with a ratio of Mg(II)/ATP > 2. Substrate Km values at 70 degrees C differ for EI versus EII but are quite comparable to those seen for mesophilic glutamine synthetases. Studies with structural analogs of substrates indicate that active site specificity is maintained at extreme temperatures: substitution of alpha-OH for alpha-HN2 is allowed, but unfavorable changes occur upon substitution of methyl groups for the alpha-H or onto the alpha-NH2 of L-Glu, and for D-Glu or L-Asp. EII is almost absdolutely specific for ATP, but EI can also use ITP, GTP, and UTP as substrates to some extent. The divalent metal ion that is present can affect both specificity for analogs and substrate Km values. Kinetic binding plots (v versus [S]) are biphasic for NH3 and L-Glu with the more active forms of each enzyme, EI-Mg and EII-Mn, respectively; but no positive cooperativity is observed. ATP binding is strictly hyperbolic, in contrast to the positive cooperativity previously observed with other Bacillus sp. enzymes. For purified EI and EII, Arrhenius plots are nonlinear with Mn(II) or Mg(II), exhibiting slope changes in the range of 55-65 degrees C; however, for EI-EII mixtures in crude cell extracts these plots are nearly linear.     

Thermine: A new polyamine from an extreme thermophile

A new polyamine compound was isolated from an extreme thermophile, $\text{Thermus thermophilus}$ HB8, and named thermine. Chemical structure of thermine was determined to be 1,11-diamino-4,8-diazaundecane, NH₂CH₂CH₂CH₂NHCH₂CH₂CH₂NHCH₂CH₂CH₂NH₂.     

Production and partial characterization of thermostable and calcium-independent alpha-amylase of an extreme thermophile Bacillus thermooleovorans NP54

AIM: An investigation was carried out on the production of alpha-amylase by Bacillus thermooleovorans NP54, its partial purification and characterization. METHODS AND RESULTS: The thermophilic bacterium was grown in shake flasks and a laboratory fermenter containing 2% soluble starch, 0.3% tryptone, 0.3% yeast extract and 0.1% K2HPO4 at 70 degrees C and pH 7.0, agitated at 200 rev min(-1) with 6-h-old inoculum (2% v/v) for 12 h. When the enzyme was partially purified using acetone (80%[v/v] saturation), a 43.7% recovery of enzyme with 6.2-fold purification was recorded. The KM and Vmax (soluble starch) values were 0.83 mg ml(-1) and 250 micromol mg(-1) protein min(-1), respectively. The enzyme was optimally active at 100 degrees C and pH 8.0 with a half-life of 3 h at 100 degrees C. Both alpha-amylase activity and production were Ca2+ independent. CONCLUSIONS: Bacillus thermooleovorans NP54 produced calcium-independent and thermostable alpha-amylase. SIGNIFICANCE AND IMPACT OF THE STUDY: The calcium-independent and thermostable alpha-amylase of B. thermooleovorans NP54 will be extremely useful in starch saccharification since the alpha-amylases used in the starch industry are calcium dependent. The use of this enzyme in starch hydrolysis eliminates the use of calcium in starch liquefaction and subsequent removal by ion exchange.     

Purification and some properties of cytochrome c-552 from an extreme thermophile, Thermus thermophilus HB8.

A c-type cytochrome, cytochrome c-552, from a soluble fraction of an extreme thermophile, Thermus thermophilus HB8, was highly purified and its properties investigated. The absorption peaks were at 552, 522, and 417 nm in the reduced form, and at 408 nm in the oxidized form. The isoelectric point was at PH 10.8, the midpoint redox potential was about +0.23 V, and the molecular weight was about 15,000. The cytochrome c-552 was highly thermoresistant. The cytochrome reacted rapidly with pseudomonas aeruginosa nitrite reductase [EC 1.9.3.2], but slowly with bovine cytochrome oxidase [EC 1.9.3.1], yeast cytochrome c peroxidase [EC 1.11.1.5], or Nitrosomonas europaea hydroxylamine-cytochrome c reductase [EC 1.7.3.4].     

Sequence structure and expression of a cloned beta-glucosidase gene from an extreme thermophile

Summary The gene for a -glucosidase from the extremely thermophilic bacterium Caldocellum saccharolyticum has been isolated from a genomic library and sequenced. An open reading frame identified by computer analysis of the sequence could encode a protein of M_r 54400, which is close to the size of the polypeptide experimentally determined using maxicells. Analysis of the amino-terminal residues of the protein produced in Escherichia coli suggests that it is processed by a methionine aminopeptidase. A sequence within C. saccharolyticum DNA upstream of the -glucosidase gene was found to act as a promoter for expression of the thermophile gene in E. coli. The protein has been overproduced in E. coli and Bacillus subtilis where it retains its enzymatic activity and heat stability. There appears to be a single copy of the gene in Caldocellum DNA.