DBF (Disulfide Bond Forming) Enzyme from the Hyperthermophilic Archaebacterium Sulfolobus Solfataricus Behaves Like a Molecular Chaperone

DBF enzyme from the hyperthermophilic archaebacterium Sulfolobus solfataricus greatly enhances the refolding at 30°C of denatured and reduced bovine pancreatic ribonuclease (Guagliardi et al., 1992). Here we show that DBF behaves like a molecular chaperone: it affects in an ATP-dependent manner the in vitro refolding at 50°C of two thermostable dehydrogenases, an alcohol dehydrogenase and a glutamate dehydrogenase from S. solfataricus. This paper also reports the complete amino acid sequence of DBF. The role of molecular chaperones from thermophilic microorganisms in applied biocatalysis is discussed.     

Effects of Temperature on Stereochemistry of Enzymatic Reactions

A brief discussion of the theoretical basis for effects of temperature on stereoselectivity of enzyme catalysed reactions is presented. In theory, the stereoselectivity of an enzymatic reaction can either increase or decrease as the reaction temperature is raised. The secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus reduces 2-butanone to (R)-2-butanol at 37° C, with increased stereoselectivity at higher temperatures and in the presence of NADP analogues. In contrast, at 37°, 2-pentanone and 2-hexanone are reduced to (S)-2-pentanol and (S)-2-hexanol, respectively, but the stereoselectivity decreases at higher temperatures and in the presence of NADP analogues. Reduction of racemic 2-methylbutanal by the primary alcohol dehydrogenase from T. ethanolicus gives (S)-2-methyl-1-butanol with greater stereospecificity at 35° (51% e.e.) than at 15° (14% e.e.). Horse liver alcohol dehydrogenase shows a preference for oxidation of the (S)-enantiomers of acyclic secondary alcohols at 25°, with a decrease in stereospecificity at higher temperatures.     

Isolation and characterization of a metagenome-derived and cold-active lipase with high stereospecificity for (R)-ibuprofen esters

We report on the isolation and biochemical characterization of a novel, cold-active and metagenome-derived lipase with a high stereo-selectivity for pharmaceutically important substrates. The respective gene was isolated from a cosmid library derived from oil contaminated soil and designated lipCE. The deduced aa sequence indicates that the protein belongs to the lipase family l.3, with high similarity to Pseudomonas fluorescens lipases containing a C-terminal secretion signal for ABC dependent transport together with possible motifs for Ca²⁺-binding sites. The overexpressed protein revealed a molecular weight of 53.2 kDa and was purified by refolding from inclusion bodies after expression in Escherichia coli. The optimum temperature of LipCE was determined to be 30 °C. However, the enzyme still displayed 28% residual activity at 0 °C and 16% at −5 °C. Calcium ions strongly increased activity and thermal stability of the protein. Further detailed biochemical characterization of the recombinant enzyme showed an optimum pH of 7 and that it retained activity in the presence of a range of metal ions and solvents. A detailed analysis of the enzyme's substrate spectrum with more than 34 different substrates indicated that the enzyme was able to hydrolyze a wide variety of substrates including the conversion of long chain fatty acid substrates with maximum activity for pNP-caprate (C₁₀). Furthermore LipCE was able to hydrolyze stereo-selectively ibuprofen-pNP ester with a high preference for the (R) enantiomer of >91% ee and it demonstrated selectivity for esters of primary alcohols, whereas esters of secondary or tertiary alcohols were nearly not converted.     

Characterization of two extracellular proteinases from Aspergillus terreus and their role in the formation of low molecular weight endoglucanases

Two extracellular proteolytic activities from the wood degrading fungus Aspergillus terreus have been characterized. Proteinase I (serine thiol-dependent enzyme) was active over a broad pH range (7·0–10·0) and at 55°C. The second proteinase (metalloproteinase) showed optimal activity at pH 6·0–7·0 and at 65–70°C. Both proteins had isoelectric points at acid pH and contained carbohydrate moieties. The metalloproteinase possessed a uniquely high content of serine and threonine and an extremely low percentage of glutamate and aspartate. The metalloproteinase was involved in the formation of the low molecular mass endoglucanases of A. terreus.     

Inactivation of complex I of the respiratory chain of maize mitochondria incubated in vitro by elevated temperature

The functional stability of the ‘external’ NADH dehydrogenase and complexes I–IV of the respiratory chain of maize mitochondria was studied during mitochondria incubation in vitro at elevated temperatures. The increase in the incubation temperature from 0°C to 37°C significantly changed the stability of the respiratory chain. At 27°C and higher, the rate of oxidation of NAD-depended substrates decreased drastically, which is related to inactivation of complex I. Complexes II, III and IV of the respiratory chain and the ‘external’ NADH dehydrogenase were functionally stable at elevated temperatures. Moreover, the possibility of electron transport during oxidation of NAD-dependent substrates, in particular malate, bypasses complex I using rotenon insensitive NADH dehydrogenase.     

Thermostable glutamate dehydrogenase from a commensal thermophile, Symbiobacterium toebii; overproduction, characterization, and application

A gene encoding glutamate dehydrogenase (GDH) was found in the genome sequence of a commensal thermophile, Symbiobacterium toebii. The amino acid sequence deduced from the gdh I of S. toebii was well conserved with other thermostable GDHs. The gdh I which encodes GDH consisting of 409 amino acids was cloned and expressed in E. coli DH5 under the control of a highly constitutive expression (HCE) promoter in a pHCE system. The recombinant GDH was expressed without addition of any inducers in a soluble form. The molecular mass of the GDH was estimated to be 263 kDa by Superose 6 HR gel filtration chromatography and 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicating that the GDH was composed of hexameric form. The optimal temperature and pH of the purified enzyme were 60 °C and 9.0, respectively, and the purified GDH retained more than 75% of its original activity after an incubation at 70 °C for 30 min. Although NADP(H) was the preferred cofactor, S. toebii GDH was able to utilize either NADP(H) or NAD(H) as coenzyme.     

A thermostable alkaline active endo-β-1-4-xylanase from Bacillus halodurans S7: Purification and characterization

A thermostable, alkaline active xylanase was purified to homogeneity from the culture supernatant of an alkaliphilic Bacillus halodurans S7, which was isolated from a soda lake in the Ethiopian Rift Valley. The molecular weight and the pI of this enzyme were estimated to be around 43 kDa and 4.5, respectively. When assayed at 70 °C, it was optimally active at pH 9.0–9.5. The optimum temperature for the activity was 75 °C at pH 9 and 70 °C at pH 10. The enzyme was stable over a broad pH range and showed good thermal stability when incubated at 65 °C in pH 9 buffer. The enzyme activity was strongly inhibited by Mn²⁺. Partial inhibition was also observed in the presence of 5 mM Cu²⁺, Co²⁺ and EDTA. Inhibition by Hg²⁺ and dithiothreitol was insignificant. The enzyme was free from cellulase activity and degraded xylan in an endo-fashion.     

Effect of nifedipine on core cooling in rats during tail cold water immersion

Male rats (450 g, n=11/group) were heated at an ambient temperature of 42°C until a rectal temperature of 42.8°C was attained. Rats, then received either saline (30°C)+tail ice water immersion (F+I) or saline (30°C)+tail ice water immersion+Nifedipine, a peripheral vasodilator, (F+I+N) to determine cooling rate effectiveness and survivability. The time to reach a rectal temperature of 42.8°C averaged 172 min in both groups resulting in similar heating rates (0.029°C/min). The cooling rates in group F+I and F+I+N were not significantly different from each other. We conclude that since Nifedipine did not improve cooling rates when combined with fluid+tail ice water immersion, its use as a cooling adjunct does not seem warranted.     

Determination of the maximum and minimum lethal temperatures (LT50) for Loxosceles intermedia Mello-Leitão, 1934 and L. laeta (Nicolet, 1849) (Araneae, Sicariidae)

In this study, the maximum and minimum lethal temperatures (LT₅₀) of L. intermedia and L. laeta were determined in two treatments: gradual heating (25–50°C) and cooling (25°C to −5°C), and 1 h at a constant temperature. In gradual temperatures change, L. intermedia mortality started at 40°C and the LT₅₀ was 42°C; for L. laeta, mortality began at 35°C and the LT₅₀ was 40°C. At low temperatures, mortality was registered only at −5°C for both species. In the constant temperature L. intermedia showed a maximum LT₅₀ at 35°C and L. laeta at 32°C; the minimum LT for both species was −7°C.     

Lack of UV-induced respiration shutoff in a recF strain of Escherichia coli: temperature conditional suppression at 30 degrees C by the sfrA mutation

A mutation in the recF gene of Escherichia coli results in a radiation-sensitive strain. The RecF pathway and the RecBC pathway account for nearly all of the conjugative recombination occuring in E. coli. recBC cells are radiation-sensitive and carry only out a small amount of recombination but these deficiencies are suppressed by an sbcB as recombination is shunted to the RecF pathway. A recBC sbcB recF strain is very radiation-sensitive and is devoid of recombination ability. These deficiencies are suppressed by the srfA mutation; srfA is a recA allele. UV-induced respiration shutoff is a recA⁺, lexA⁺ and recBC⁺ dependent. We report in this paper that respiration does not shutoff in a recF strain at 37 and 30°C. an srfA mutation suppresses this lack of respiration shutoff effect in a recF srfA mutant at 30°C but not at 37°C; no suppression by this mutation occurs at either temperature in a recF recBC sbcB strain. An srfA strain also does not shut off its respiration at 37°C and shows a temperature conditional UV-induced respiration shutoff response at 30°C. The srfA mutation is thought to cause an altered RecA protein to be produced and we suggest that at 37° This altered protein is temperature sensitive. We conclude from the results in this paper that the recF gene product is required for UV-induced respiration shutoff and that the RecA protein plays a special role in the induction process.     

Temperature dependence of the germination of tomato seeds

1. 1.|The germination of tomato “C38” seeds exposed to periodical white incandescent light occurs from 6.0° ± 0.2°C to 37.5° ± 0.2°C, being rate-limited for 10.3° T 25.9°C, and elsewhere limited by the germination capacity.

2. 2.|Rate averages are linearly T-dependent outside their optimum range (25.9° T 29.5°C) and rate variances are typically heterogeneous.

3. 3.|The smooth curvilinear Arrhenius plot indicates that diffusion processes cannot be rate-limiting outside the interval 25.9° T 29.5°C, whereas phase transitions and (or) transconformation of proteins may limit the rate above 34.9°C and, by opposite effects, below 15.3°C.

4. 4.|The thermal communication between the environment and the germinating seed proceeds by a temperature signal which is quenched by random thermal noise at T 11.2°C and at T 34.0°C.

Effect of combination treatment with entomopathogenic fungi Beauveria bassiana and Nomuraea rileyi (Hypocreales) on Spodoptera litura (Lepidoptera: Noctuidaeae)

In biocontrol of insect pests, efficacy of treatment with multiple pathogens has not been frequently investigated but may have potential for effective management. The possible advantage of a combination treatment with two entomopathogenic fungi - Beauveria bassiana and Nomuraea rileyi - was assessed in laboratory bioassays on second instar Spodoptera litura. From among the fungal isolates of an epizootic population, two isolates of each fungus differing in virulence to S. litura were chosen, one highly virulent and the other with low virulence. The bioassays were carried out at either a continuous temperature of 25±1°C or at a temperature cycle of 32±2°C 8 h/21±2°C 16 h to mimic the field temperatures during the epizootic. Treatments with the two fungi were done both simultaneously and sequentially. In combination treatments at 25±1°C, in all isolate combinations, a majority of the larvae showed N. rileyi induced mycosis; the percentage mortality and speed of kill of insects in these treatments was similar to the N. rileyi isolate used in the combination treatments. At the temperature cycle of 32±2°C 8 h/21±2°C 16 h, in all combination treatments, all the dead insects exhibited B. bassiana mycosis; the mortality pattern was similar to the B. bassiana isolate used in the combination treatments. The adverse effect of high temperature on virulence of N. rileyi was however, not evident in in vitro growth assays. Combination treatment with both fungi did not have a synergistic effect on insect mortality.     

Thermal avoidance and preference in Daphnia magna

1. Water fleas (Daphnia magna) bred at 23°C were non-responsive to temperatures between 13 and 25°C.

2. At the lower (11°C) and upper limits (30°C) their klinokinetic avoidance behaviour showed a larger intraindividual than interindividual variation.

3. Thermal sensitivity for avoidance responses in D. magna was about 1.5°C.

4. For D. magna bred for one parthenogenetic generation at 14°C heat avoidance temperature was about 8°C lower, and cold avoidance temperature was about 1°C higher than in D. magna from 23°C.

5. In group experiments the animals showed some preference for the acclimation temperature.

6. Cold induced stenothermy and warm induced eurythermy in D. magna were related to the mode of reproduction.

Enzyme stabilization using synthetic compensatory solutes

The protective effect of the synthetic compensatory solutes, dimethylthetin (CAS 4727-41-7) and homodeanol betaine (N, N-dimethyl-N-(2-hydroxyethyl)-N-(2 carboxyethyl) ammonium inner salt, CAS 6249-53-2), on two enzymes: lactate dehydrogenase (LDH from rabbit muscle) and a microbial lipase, was compared with that of glycine betaine, trehalose and sorbitol. When the enzyme plus 1 M solute were heated for 10 min at temperatures between 35-75°C, the temperature at which 50% of enzyme activity was lost increased most in the presence of trehalose (7.9° for LDH, 11.6° for lipase) and homodeanol betaine (10.7° for LDH, 11.0° for lipase). With both enzymes, more activity was retained at extreme temperatures in the presence of homodeanol betaine than with trehalose. Glycine betaine, dimethylthetin and sorbitol were less effective. Enzyme plus 1 M stabilizer solutions were frozen at -30°C and freeze-dried for 24 h. Trehalose was the most effective stabilizer of lactate dehydrogenase, and homodeanol betaine of lipase, during freeze-drying.     

The Sulfolobus tokodaii gene ST1704 codes highly thermostable glucose dehydrogenase

NAD(P)-dependent glucose-1-dehydrogenase (GDH) has been used for glucose determination and NAD(P)H production in bioreactors. Thermostable glucose dehydrogenase exhibits potential advantage for its application in biological processes. The function of the putative GDH gene (ST1704, 360-encoding amino acids) annotated from the total genome analysis of a thermoacidophilic archeaon Sulfolobus tokodaii strain 7 was investigated to develop more effective application of GDH. The gene encoding S. tokodaii GDH was cloned and the activity was expressed in Escherichia coli, which did not originally possess GDH. This shows that the gene (ST1704) codes the sequence of GDH. The enzyme was effectively purified from the recombinant E. coli with three steps containing a heat treatment and two successive chromatographies. The native enzyme (molecular mass: 160 kDa) is composed of a tetrameric structure with a type of subunit (41 kDa). The enzyme utilized both NAD and NADP as the coenzyme. The maximum activity for glucose oxidation in the presence of NAD was observed around pH 9 and 75 °C in the presence of 20 mM Mg²⁺. The enzyme showed broad substrate specificity: several monosaccarides such as 6-deoxy- -glucose, 2-amino-2-deoxy- -glucose and -xylose were oxidized as well as -glucose as the electron donor. -Mannose, -ribose and glucose-6-phosphate were inert as the donor. The enzyme showed high thermostability: remarkable loss of activity was not observed up to 80 °C by incubation for 15 min at pH 8.0. In addition, the enzyme was stable in a wide pH range of 5.0–10.5 by incubation at 37 °C. From the steady-state kinetic analysis, the enzyme reaction of -glucose oxidation proceeds via a sequential ordered Bi–Bi mechanism: NAD and -glucose bind to the enzyme in this order and then -glucono-1,5-lactone and NADH are released from the enzyme in this order. The amino acid sequence alignment showed that S. tokodaii GDH exhibited high homology with the Sulfolobus solfataricus hypothetical glucose dehydrogenase and a Thermoplasma acidophilum one.     

Production, purification and characterization of an endopolygalacturonase from Mucor rouxii NRRL 1894

An extracellular polygalacturonase (PGase) from Mucor rouxii NRRL 1894 was purified to homogeneity by two chromatographic steps using CM-Sepharose and Superdex 75. The purified enzyme was a monomer with a molecular weight of 43100 Da and a pI of 6. The PGase was optimally active at 35 °C and at pH 4.5. It was stable up to 30 °C and stability of PGase decrease rapidly above 60 °C. The extent of hydrolysis of different pectins was decreased with increasing of degrees of esterification. Except Mn²⁺, all the examined metal cations showed inhibitory effects on the enzyme activity. The apparent K_m and V_max values for hydrolyze of polygalacturonic acid (PGA) were 1.88 mg/ml and 0.045 μmol/ml/min, respectively. The enzyme released a series of oligogalacturonates from polygalacturonic acid indicating that it had an endo-action. Its N-terminal sequence showed homologies with the endopolygalacturonase from the psychrophilic fungus Mucor flavus.     

The effect of temperature on the duration of the egg stage of certain sciomyzid flies which predate Lymnae truncatula

1. 1. The effect of temperature on the duration of the egg stage of 3 species of sciomyzid flies was investigated at 14, 17, 20, 23 and 26°C.

2. 2. Ilione albiseta(Scopoli): the mean duration of the egg stage (m.d.e.s) decreased from 88.53 to 34.05 days as temperature increased up to 26°C. Under changing temperature conditions (14↔20°C) there was a significant retardation in the duration of the egg stage. 50–70% of conditioned embryonated eggs (i.e. eggs maintained on moist filter paper from 10 to 20 days at 23°C) hatched within 24 in a 0.1% solution of ascorbic acid. (Embryonation was judged to have taken place when the cephalopharyngeal skeleton was visible through the chorion and there was evidence of larval movement within the egg.)

3. 3. Limnia unguicornis (Scopoli): the m.d.e.s. at the 5 constant temperatures was very variable but was significantly shorter at 26°C (22.85 days) than at 14 and 17°C (29.8 and 32.48 days respectively).

4. 4. Pherbellia cinerella (Fallén): the m.d.e.s. decreased from 14.73 days at 14°C to 4.42 days to 26°C and percentage hatch was greatest at 14°C.

Activity and stability of native and modified alanine aminotransferase in cosolvent systems and denaturants

Alanine aminotransferase (ALT) is used in clinical diagnostics, amino acid synthesis and in biosensors. Here we describe the stabilization of soluble porcine ALT by chemical modification with mono- and bis-imidates. The apparent transition temperatures (‘T_m’, the temperature where 50% of initial activity was lost in 10 min) for native and DMS-modified ALT were 46 and 56 °C respectively. The effects of water-miscible organic solvents (methanol, dimethylformamide, dimethylsulphoxide and 1,4-dioxane) on the activity/stability of native and modified forms were determined. In all systems studied, an abrupt decrease in ALT catalytic activity was observed on reaching a certain threshold concentration of the organic solvent. The modified derivatives were more organotolerant than native enzyme. Comparison of the apparent V_max and K_m for 2-oxoglutarate as substrate, determined in 10% (v/v) organic solvent, with the results of thermal inactivation studies showed that the solvents have different effects on ALT's catalytic parameters and on its conformational stability. At 35 °C with no organic solvent the dimethylsuberimidate (DMS)-modified derivative's half-life was 16 times greater than that for native enzyme; in 30% (v/v) solvent at 35 °C, the DMS-modified ALT's half-life was up to 4.6 times greater than native enzyme's. DMS-modified ALT was also more stable in urea and guanidine HCl, and its refolding was more noticeable, than that of native enzyme.     

A nicotinamide mononucleotide adenylyltransferase with unique adenylyl group donor specificity from a hyperthermophilic archaeon, Pyrococcus horikoshii OT-3

A gene encoding a nicotinamide mononucleotide (NMN) adenylyltransferase (NMNAT, EC 2.7.7.1) homologue was identified via genome sequencing in the anaerobic hyperthermophilic archaeon Pyrococcus horikoshii OT-3. The gene encoded a protein of 186 amino acids with a molecular weight of 21,391. The deduced amino acid sequence of the gene showed 59% identities to the NMNAT from Methanococcus jannaschii. The gene was overexpressed in Escherichia coli, and the produced enzyme was purified to homogeneity. Characterization of the enzyme revealed that it is an extremely thermostable NMNAT; the activity was not lost after incubation at 80 °C for 30 min. The native molecular mass was estimated to be 77 kDa. The K_m values for ATP and NMN were calculated to be 0.056 and 0.061 mM, respectively. The optimum temperature of the reaction was estimated to be around 90 °C. The adenylyl group donor specificity was examined by high-performance liquid chromatography (HPLC). At 70 °C, ATP was a prominent donor. However, above 80 °C, a relatively small, but significant, NMNAT activity was detected when ATP was replaced by ADP or AMP in the reaction mixture. To date, an NMNAT that utilizes ADP or AMP as an adenylyl group donor has not been found. The present study provides interesting information in which a di- or mono-phosphate nucleotide can be utilized by adenylyltransferase at high temperature.