Roy M. Daniel

Fractionation of nitrogen isotopes by animals: a further complication to the use of variations in the natural abundance of 15N for tracer studies

A study of the fractionation of nitrogen isotopes in the diet by cattle is described and the results discussed. Compared with the diet, urine had a lower ratio of 15 N to 14 N, but faeces, blood and milk all had a higher ratio. It is argued that the use of natural 15 N as a tracer in grazed ecosystems is more complicated than was at first thought.

Detection and impact of protease and lipase activities in milk and milk powders

Freshly drawn milk contains indigenous enzymes, including proteases and lipases. During handling and processing, milk acquires contaminating bacteria that produce further proteases and lipases, adding to the enzyme loading in the milk. Even when several heat treatment steps are used to prepare milk products, these will not be sufficient to inactivate all of the enzymes. Adverse effects of heat on the product limit the extent of heat treatments that can be used. The activities of proteinases and lipases that survive the heat treatments may cause changes in functionality and flavour of milk products, including milk powders, during storage. This review focuses on the characteristics of proteases (specifically proteinases) and lipases in milk and milk powders, detection methods for such enzymes and the effects of these enzymes on milk and milk powders during storage.

A Hydrothermally Precipitated Catalytic Iron Sulphide Membrane as a First Step Toward Life

We propose that life emerged from growing aggregates of iron sulphide bubbles containing alkaline and highly reduced hydrothermal solution. These bubbles were inflated hydrostatically at sulphidic submarine hot springs sited some distance from oceanic spreading centers four billion years ago. The membrane enclosing the bubbles was precipitated in response to contact between the spring waters and the mildly oxidized, acidic and iron-bearing Hadean ocean water. As the gelatinous sulphide bubbles aged and were inflated beyond their strength they budded, producing contiguous daughter bubbles by the precipitation of new membrane. [Fe2S2]+/0 or [Fe4S4]2+/+ clusters, possibly bonded by hydrothermal thiolate ligands as proferredoxins, could have catalyzed oxidation of thiolates to disulphides, thereby modifying membrane properties.We envisage the earliest iron sulphide bubbles (pro botryoids) first growing by hydrostatic inflation with hydrothermal fluid, but evolving to grow mainly by osmosis (the protocellular stage), driven by (1) catabolism of hydrothermal abiogenic organics trapped on the inner walls of the membrane, catalyzed by the iron sulphide clusters; and (2) cleavage of hydrophobic compounds dissolved in the membrane to hydrophilic moieties which were translocated, by the proton motive force inherent in the acidic Hadean ocean, to the alkaline interior of the protocell. The organics were generated first within the hydrothermal convective system feeding the hot springs operating in the oceanic crust and later in the pyritizing mound developing on the sea floor, as a consequence of the reduction of CO, CO2, and formaldehyde by Fe2+- and S2−-bearing minerals.We imagine the physicochemical interactions in and on the membrane to have been sufficiently complex to have engendered auto- and cross-catalytic replication. The membrane may have been constructed in such a way that a “successful” parent could have “informed” the daughters of membrane characteristics functional for the then-current level of evolution.

Biomolecular stability and life at high temperatures

Abstract. It is not clear what the upper temperature limit forlife is, or what specific factors will set this limit, but it is generally assumed that the limit will be dictated by molecular instability. In this review, we examine the thermal stability of two key groups of biological molecules: the intracellular small molecules/metabolites and the major classes of macromolecules. Certain small molecules/metabolites are unstable in vitro at the growth temperatures of the hyperthermophiles in which they are found. This instability appears to be dealt with in vivo by a range of mechanisms including rapid turnover, metabolic channelling and local stabilisation. Evidence to date suggests that proteins have the potential to be stable at substantially higher temperatures than those known to support life, but evidence concerning degradative reactions above 100 °C is slight. DNA duplex stability is apparently achieved at high temperature by elevated salt concentrations, polyamines, cationic proteins, and supercoiling rather than manipulation of C-G ratios. RNA stability seems dependent upon covalent modification, although secondary structure is probably also critical. The diether-linked lipids, which make up the monolayer membrane of most organisms growing above 85 °C are chemically very stable and seem potentially capable of maintaining membrane integrity at much higher temperatures. However, the in vivo implications of the in vitro instability of biomolecules are difficult to assess, and in vivo data are rare.

The upper limits of enzyme thermal stability

Abstract Some enzymes, such as those from extreme thermophiles, have significant half-lives above 100°C. The differences in structure and function between these very stable and less stable enzymes are relatively small and are comparable with those differences found among enzymes of similar stability. Recent evidence suggests that protein degradative reactions at high temperatures (>80°C) occur only slowly in conformationally intact proteins, so that conformational stability may still dictate the upper temperature limit for enzyme activity. The interrelationship of both conformational stability and enzymatic activity with protein flexibility suggests that in naturally occurring enzymes, we cannot expect to find stability at temperatures far above those which are optimum for the growth of the organism. Genetic and enzyme engineering studies are promising in terms of enhancing conformational stability, but are likely to require case-by-case knowledge of the enzyme concerned, and stability enhancements achieved so far are relatively small. Furthermore, engineered increases in stability may well be accompanied by a decrease in specific activity. Nevertheless, it should be feasible to engineer enzymes to be substantially more stable than any of those found so far in nature where useful half-lives above 120°C are already available.

Enzyme Activity below the Dynamical Transition at 220 K

Enzyme activity requires the activation of anharmonic motions, such as jumps between potential energy wells. However, in general, the forms and time scales of the functionally important anharmonic dynamics coupled to motion along the reaction coordinate remain to be determined. In particular, the question arises whether the temperature-dependent dynamical transition from harmonic to anharmonic motion in proteins, which has been observed experimentally and using molecular dynamics simulation, involves the activation of motions required for enzyme function. Here we present parallel measurements of the activity and dynamics of a cryosolution of glutamate dehydrogenase as a function of temperature. The dynamical atomic fluctuations faster than approximately 100 ps were determined using neutron scattering. The results show that the enzyme remains active below the dynamical transition observed at approximately 220 K, i.e., at temperatures where no anharmonic motion is detected. Furthermore, the activity shows no significant deviation from Arrhenius behavior down to 190 K. The results indicate that the observed transition in the enzymes dynamics is decoupled from the rate-limiting step along the reaction coordinate.

Very stable enzymes from extremely thermophilic archaebacteria and eubacteria

SummaryThirty-six thermophilic archaebacteria and nine extremely thermophilic eubacteria have been screened on solid media for extracellular amylase, protease, hemicellulase (xylanase), cellulase, pectinase and lipase activities. Extracellular enzymes were detected in 14 archaebacteria belonging to three different orders. Twelve of these were able to degrade starch and casein and the two Thermofilum strains were able to degrade starch, xylan and carboxymethylcellulose. Three of the eubacteria could degrade only starch. The other six (including four Thermotoga strains) all had activity against starch, xylan and carboxymethylcellulose, and one also had activity against casein. Some of the amylolytic archaebacteria released α-glucosidase, β-glucosidase, amylase and transglucosylase activities into liquid media containing starch or maltose. Thermotoga strain FjSS3B.1 released amylase, xylanase, cellulase and β-glucosidase activities into the medium when grown in the presence of substrates. When the partially purified enzymes from Thermotoga and some of the archaebacteria were compared with known thermostable enzymes the majority were found to be the most thermostable of their type. The β-glucosidase, xylanase and cellulase from Thermotoga and two α-glucosidases, a β-glucosidase, an amylase and a pullulanase from archaebacteria all have half-lives of at least 15 min at 105°C.

Nitrate dependent anaerobic acetylene-reduction and nitrogen-fixation by soybean bacteroids

SUMMARY: Bacteroids isolated from nodules produced by one strain of Rhizobium japonicum (CC705) had strong nitrate-reducing activity and reduced C2H2 to C2H4 and N2 to NH3 anaerobically with nitrate. Bacteroids of another strain (CB1809) were much less active nitrate reducers and reduced little C2H2 anaerobically. Nitrite, which accumulated in the medium in anaerobic assays, was an inhibitor of C2H2 reduction in both aerobic and anaerobic conditions. Succinate, at about 25 mM, stimulated both nitrate reduction and C2H2 reduction under aerobic conditions. Glucose stimulated C2H2 reduction up to 120 mM but nitrate reduction was inhibited in the presence of glucose. In terms of electrons transferred, the aerobic pathway appeared to be about 2.5 times more efficient than the anaerobic pathway in supporting nitrogenase activity of CC705 bacteroids.

The dependence of enzyme activity on temperature: determination and validation of parameters

Traditionally, the dependence of enzyme activity on temperature has been described by a model consisting of two processes: the catalytic reaction defined by DeltaG(Dagger)(cat), and irreversible inactivation defined by DeltaG(Dagger)(inact). However, such a model does not account for the observed temperature-dependent behaviour of enzymes, and a new model has been developed and validated. This model (the Equilibrium Model) describes a new mechanism by which enzymes lose activity at high temperatures, by including an inactive form of the enzyme (E(inact)) that is in reversible equilibrium with the active form (E(act)); it is the inactive form that undergoes irreversible thermal inactivation to the thermally denatured state. This equilibrium is described by an equilibrium constant whose temperature-dependence is characterized in terms of the enthalpy of the equilibrium, DeltaH(eq), and a new thermal parameter, T(eq), which is the temperature at which the concentrations of E(act) and E(inact) are equal; T(eq) may therefore be regarded as the thermal equivalent of K(m). Characterization of an enzyme with respect to its temperature-dependent behaviour must therefore include a determination of these intrinsic properties. The Equilibrium Model has major implications for enzymology, biotechnology and understanding the evolution of enzymes. The present study presents a new direct data-fitting method based on fitting progress curves directly to the Equilibrium Model, and assesses the robustness of this procedure and the effect of assay data on the accurate determination of T(eq) and its associated parameters. It also describes simpler experimental methods for their determination than have been previously available, including those required for the application of the Equilibrium Model to non-ideal enzyme reactions.

A Numerical Classification of Some Thermus Isolates

Summary: A numerical classification was performed on a collection of 45 Thermus isolates recovered from New Zealand hot pools and on six type strains including T. aquaticus and “T. thermophilus”. Unweighted average linking (UPGMA) and single linkage clustering methods were applied to similarity matrices derived from simple matching (SSM ) and Jaccard similarity (SJ ) coefficients. Differences were observed between phenograms derived from SSM and SJ coefficients, indicating that some of the clusters formed were derived from a significant component of negative matches. Test error was estimated at 2.9%. In the UPGMA/SSM phenogram, seven clusters were formed. A majority of the New Zealand isolates did not cluster with non-New Zealand isolates. Analysis of variance showed that there was a relationship between the composition of the clusters and the temperature and pH of the source of the isolate. Chi-squared testing showed that, within New Zealand, the geographical source of the isolate had no bearing on the clusters formed.