Growth of and trehalase activity in the thermophilic fungusThermomyces lanuginosus

The thermophilic fungus,Thermomyces lanuginosus, was grown in a glucose-asparagine liquid medium. Optimal mycelial growth occurred at 50°C. The conditions for sporulation were different from those required for vegetative growth. the former being favoured by lower nitrogen level and temperature. Trehalase (α, α-glu coside-l-glucohydrolase, EC 3.2.1.28) was one of the most active glycosidases at 50°C. Non-sporulating mycelium had higher levels of this enzyme than the sporulating mycelium. Trehalase was synthesized constitutively and its activity appears to be controlled by catabolite repression.

Purification and properties of trehalase from the thermophilic fungus Humicola lanuginosa

Trehalase (?,?-Trehalosee gludohydrolase, EC 3.2.1.28) was partially solubilized from the thermophilic fungus Humicola lanuginosa RM-B, and purified 184-fold. The purified enzyme was optimally active at 50°C in acetate buffer at pH 5.5. It was highly specific for ?,?-trehalose and had an apparent Km = 0.4 mM at 50°C. None of the other disaccharides tested either inhibited or activated the enzyme. The molecular weight of the enzyme was around 170000. Trehalase from mycelium grown at 40 and 50°C had similar properties. The purified enzyme, in contrast to that in the crude-cell free extract, was less stable. At low concentration, purified trehalase was afforded protection against heat-inactivation by �protective factor(s)� present in mycelial extracts. The �protective factor(s)� was sensitive to proteolytic digestion. It was not diffusable and was stable to boiling for at least 30 min. Bovine serum albumin and casein also protected the enzyme from heat-inactivation.

Effect of Temperature on Respiration of a Mesophilic and a Thermophilic Fungus

The respiratory rates of mycelia of the mesophilic fungus, Aspergillus niger, and the thermophilic fungus, Thermomyces lanuginosus, were comparable at their respective temperature optima for growth. The respiratory rate of A. niger was independent of changes in temperature between 15 and 40 C. The respiratory rate of T. lanuginosus increased with increase in temperature between 25 and 55 C.

Temperature response of trehalase from a mesophilic (Neurospora crassa) and a thermophilic (Thermomyces lanuginosus) fungus

The purified trehalases of the mesophilic fungus, Neurospora crassa, and the thermophilic fungus, Thermomyces lanuginosus, had similar temperature and pH optima for activity, but differed in molecular weight, electrophoretic mobility and Michaelis constant. At lower concentration, trehalases from both fungi were inactivated to similar extent at 60°C. While purified trehalase of T. lanuginosus was afforded protection against heat-inactivation by proteinaceous protective factor(s) present in mycelial extracts, by bovine serum albumin and by casein, these did not afford protection to N. crassa trehalase against heat inactivation. Both trehalases exhibited discontinuous Arrhenius plots with temperature of discontinuity at 40°C. The activation energy calculated from the slope of the Arrhenius plot was higher for the T. lanuginosus enzyme. The plots of apparent K m versus 1/T for trehalases of N. crassa and T. lanuginosus were linear from 30° to 60°C. The results show that purified trehalases of the mesophilic and the thermophilic fungus are distinct. Although, these exhibit similar thermostability of their catalytic function at low concentration, distinctive thermal stability characteristics of thermophilic enzyme become apparent at high protein concentration. This could be brought about in the cell by the enzyme itself, or by other proteins.

Integrability and singularity structure of predator‐prey system

The ‘‘extended’’ ARS (Ablowitz, Ramani, and Segur) algorithm is introduced to characterize a dynamical system as Painlevé or otherwise; to that end, it is required that the formal series—the Laurent series, logarithmic, algebraic psi series about a movable singularity—are shown to converge in the deleted neighborhood of the singularity. The determinations thus obtained are compared with those following from the α method of Painlevé. An attempt is made to relate the structure of solutions about a movable singularity with that of first integrals (when they exist). All these ideas are illustrated by a comprehensive analysis of the general two‐dimensional predator‐prey system.

Singularity structure of third-order dynamical systems .2

The singularity structure of the solutions of a general third-order system, with polynomial right-hand sides of degree less than or equal to two, is studied about a movable singular point, An algorithm for transforming the given third-order system to a third-order Briot-Bouquet system is presented, The dominant behavior of a solution of the given system near a movable singularity is used to construct a transformation that changes the given system directly to a third-order Briot-Bouquet system. The results of Horn for the third-order Briot-Bouquet system are exploited to give the complete form of the series solutions of the given third-order system; convergence of these series in a deleted neighborhood of the singularity is ensured, This algorithm is used to study the singularity structure of the solutions of the Lorenz system, the Rikitake system, the three-wave interaction problem, the Rabinovich system, the Lotka-Volterra system, and the May-Leonard system for different sets of parameter values. The proposed approach goes far beyond the ARS algorithm.

Bi-Domain Protein Tyrosine Phosphatases Reveal an Evolutionary Adaptation to Optimize Signal Transduction

Significance: The bi-domain protein tyrosine phosphatases (PTPs) exemplify functional evolution in signaling proteins for optimal spatiotemporal signal transduction. Bi-domain PTPs are products of gene duplication. The catalytic activity, however, is often localized to one PTP domain. The inactive PTP domain adopts multiple functional roles. These include modulation of catalytic activity, substrate specificity, and stability of the bi-domain enzyme. In some cases, the inactive PTP domain is a receptor for redox stimuli. Since multiple bi-domain PTPs are concurrently active in related cellular pathways, a stringent regulatory mechanism and selective cross-talk is essential to ensure fidelity in signal transduction. Recent Advances: The inactive PTP domain is an activator for the catalytic PTP domain in some cases, whereas it reduces catalytic activity in other bi-domain PTPs. The relative orientation of the two domains provides a conformational rationale for this regulatory mechanism. Recent structural and biochemical data reveal that these PTP domains participate in substrate recruitment. The inactive PTP domain has also been demonstrated to undergo substantial conformational rearrangement and oligomerization under oxidative stress. Critical Issues and Future Directions: The role of the inactive PTP domain in coupling environmental stimuli with catalytic activity needs to be further examined. Another aspect that merits attention is the role of this domain in substrate recruitment. These aspects have been poorly characterized in vivo. These lacunae currently restrict our understanding of neo-functionalization of the inactive PTP domain in the bi-domain enzyme. It appears likely that more data from these research themes could form the basis for understanding the fidelity in intracellular signal transduction.

Avoiding acidic region streaking in two-dimensional gel electrophoresis: Case study with two bacterial whole cell protein extracts

Acidic region streaking (ARS) is one of the lacunae in two-dimensional gel electrophoresis (2DE) of bacterial proteome. This streaking is primarily caused by nucleic acid (NuA) contamination and poses major problem in the downstream processes like image analysis and protein identification. Although cleanup and nuclease digestion are practiced as remedial options, these strategies may incur loss in protein recovery and perform incomplete removal of NuA. As a result, ARS has remained a common observation across publications, including the recent ones. In this work, we demonstrate how ultrasound wave can be used to shear NuA in plain ice-cooled water, facilitating the elimination of ARS in the 2DE gels without the need for any additional sample cleanup tasks. In combination with a suitable buffer recipe, IEF program and frequent paper-wick changing approach, we are able to reproducibly demonstrate the production of clean 2DE gels with improved protein recovery and negligible or no ARS. We illustrate our procedure using whole cell protein extracts from two diverse organisms, Escherichia coli and Mycobacterium smegmatis. Our designed protocols are straightforward and expected to provide good 2DE gels without ARS, with comparable times and significantly lower cost.